Human NMDA R2A receptor subunit and isoforms of the human NMDA-R1 receptor subunit and encoding cDNAs

ABSTRACT

The present invention relates to a stably co-transfected eukaryotic cell line that expresses an N-methyl-D-aspartate (NMDA) receptor, particularly a human NMDA receptor, which receptor comprises at least one R1 subunit isoform, or at least one R1 subunit isoform and one or two R2 subunits. Additionally, the cell line can be used to design and develop NMDA receptor subtype-selective compounds. The invention also relates to cloning of novel cDNA sequences encoding the human NMDAR 2A subunit and various isoforms of the human NMDA R1 subunit.

FIELD OF THE INVENTION

The present invention concerns a cell line, and in particular relates toa stable cell line capable of expressing human or animalN-methyl-D-aspartate (NMDA) receptors. The invention also relates tocomplementary DNAs (cDNAs) encoding novel human NMDA receptor subunits,and concerns in particular the nucleotide and deduced amino acidsequences of the human NMDA R2A receptor subunit, and of variousisoforms of the human NMDA R1 receptor subunit.

BACKGROUND

The NMDA receptor is the major excitatory amino acid receptor thatmediates glutamate transmission in the central nervous system. Thisreceptor has been implicated in neuronal modulation, including long termpotentiation in the hippocampus (Collingridge and Singer, TIPS, 1990,11, 290). It is consequently believed to play a key role in memoryacquisition and learning.

The integral channel of the NMDA receptor allows ca²⁺ to permeate aswell as Na⁺ and K⁺, and presents at least seven pharmacologicallydistinct sites. These include a glutamate binding site, a glycinebinding site, a polyamine site, a dizocilpine binding site, a voltagedependent Mg²⁺ site, a Zn²⁺ binding site (Wong and Kemp, Ann. Rev.Pharmacol. Toxicol., 1991, 31, 401) and an ifenprodil binding site(Carter et al., J. Pharmacol. Exy. Ther., 1988, 247, 1222).

The development of potent and selective NMDA receptor antagonists whichpenetrate into the brain has received considerable attention of late asan attractive strategy for treating and/or preventing conditions whichare believed to arise from over-stimulation of neurotransmitter releaseby excitatory amino acids. Such conditions notably includeneurodegenerative disorders arising as a consequence of suchpathological conditions as stroke, hypoglycaemia, cerebral palsy,transient cerebral ischaemic attack, cerebral ischaemia during cardiacpulmonary surgery or cardiac arrest, perinatal asphyxia, epilepsy,Huntington's chorea, Alzheimer's disease, Amyotrophic Lateral Sclerosis,Parkinson's disease, Olivo-ponto-cerebellar atrophy, anoxia such as fromdrowning, spinal cord and head injury, and poisoning by exogenous andendogenous NMDA receptor agonists and neurotoxins, includingenvironmental neurotoxins.

NMDA receptor antagonists may also be useful as anticonvulsant andantiemetic agents, as well as being of value in the prevention orreduction of dependence on dependence-inducing agents such as narcotics.

NMDA receptor antagonists have recently been shown to possess analgesic(see, for example, Dickenson and Aydar, Neuroscience Lett., 1991, 121,263; Murray et al., Pain, 1991, 44, 179; and Woolf and Thompson, Pain,1991, 44, 293) and anxiolytic (see, for example, U.S. Pat. No.5,145,866; and Kehne et al., Eur. J. Pharmacol., 1991, 193, 283)effects, and such compounds may accordingly be useful in the managementof pain, depression and anxiety.

Compounds possessing functional antagonist properties for the NMDAreceptor complex are stated in WO-A-91/19493 to be effective in thetreatment of mood disorders, including major depression, bipolardisorder, dysthymia and seasonal affective disorder (cf. also Trullasand Skolnick, Eur. J. Pharmacol., 1990, 185, 1). Such compounds mayconsequently be of benefit in the treatment and/or prevention of thosedisorders.

The association of NMDA receptor antagonists with regulation of thedopaminergic system has recently been reported (see, for example,Werling et al., J. Pharmacol. Exp. Ther., 1990, 255, 40; Graham et al.,Life Sciences, 1990, 47, PL-41; Hutson et al., Br. J. Pharmacol., 1991,103, 2037; and Turski et al., Nature (London), 1991, 349, 414). Thissuggests that such compounds may thus be of assistance in the preventionand/or treatment of disorders of the dopaminergic system such asschizophrenia and Parkinson's disease.

It has also been reported recently (see Lauritzen et al., Journal ofCerebral Blood Flow and Metabolism, 1991, vol. 11, suppl. 2, AbstractXV-4) that NMDA receptor antagonists block cortical spreading depression(CSD), which may thus be of clinical importance since CSD is a possiblemechanism of migraine. The class of substituted2-amino-4-phosphonomethylalk-3-ene carboxylic acids and esters describedin EP-A0420806, which are stated to be selective NMDA antagonists, arealleged thereby to be of potential utility in the treatment of interalia migraine.

Excitatory amino acid receptor antagonists, including inter aliaantagonists of NMDA receptors, are alleged in EP-A-0432994 to be of usein suppressing emesis.

Recent reports in the literature have also suggested a link between theneurotoxicity of certain viruses and the deleterious effects of theseviruses on an organism caused by the potentiation of neurotransmissionvia excitatory amino acid receptors. Antagonists of NMDA receptors maytherefore be effective in controlling the manifestations of neuroviraldiseases such as measles, rabies, tetanus (cf. Bagetta et al., Br. J.Pharmacol., 1990, 101, 776) and AIDS (cf. Lipton et al., Society forNeuroscience Abstracts, 1990, 16, 128.11).

NMDA antagonists have, moreover, been shown to have an effect on theneuroendocrine system (see, for example, van den Pol et al., Science,1990, 250, 1276; and Urbanski, Endocrinology, 1990, 127, 2223), and suchcompounds may therefore also be effective in the control of seasonalbreeding in mammals.

A cDNA, encoding a subunit of the rat NMDA receptor and designated NMDAR1, has been cloned by expression cloning (Moriyoshi et al., Nature(London), 1991, 354, 31). When expressed in Xenopus oocytes, this cDNAexhibits the electrophysiological and pharmacological propertiesexpected of an authentic NMDA receptor, although the levels ofexpression are extremely low. More recently, the existence of severaldiscrete isoforms of the rat NMDA R1 receptor subunit, generated byalternative RNA splicing, has been reported (Sugihara et al., BBRC,1992, 185, 826). Using both low stringency hybridization and polymerasechain reaction methodologies, four additional rodent NMDA receptorsubunit cDNAs have been cloned: ε1 or NMDA R2A; ε2 or NMDA R2B; ε3 orNMDA R2C; and ε4 or NMDA R2D (see Monyer et al., Science, 1992, 256,1217; Kutsuwada et al., Nature (London), 1992, 358, 36; Ikeda et al.,FEBS Lett., 1992, 313, 34; and Ishii et al., J. Biol. Chem., 1993, 268,2836). Co-expression in Xenopus oocytes or transiently transfected cellsof the NMDA R1 subunit, with any one of the R2A, R2B, R2C or R2Dsubunits referred to above, gives rise to a more robust NMDA receptorthan that constituted by the NMDA R1 subunit alone (Monyer et al.,Science, 1992, 256, 1217). Moreover, these four resulting putative NMDAreceptors (R1/R2A; R1/R2B; R1/R2C; and R1/R2D) are observed to bepharmacologically and electrophysiologically distinguishable. These datasupport the hypothesis that a family of NMDA receptor subtypes withdistinct pharmacological profiles may exist in the brain throughcombination of different subunits.

Any of a variety of procedures may be used to molecularly clone humanNMDA receptor cDNA. These methods include, but are not limited to,direct functional expression of the human NMDA receptor cDNAs followingthe construction of a human NMDA receptor containing cDNA library in anappropriate expression vector system. Another method is to screen ahuman NMDA receptor containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a labelled oligonucleotideprobe designed from the amino acid sequence of the purified NMDAreceptor protein or from the DNA sequence of known NMDA receptor cDNAs.The preferred method consists of screening a human NMDA receptorcontaining cDNA library constructed in a bacteriophage or plasmidshuttle vector with a ³² P-labelled cDNA oligonucleotide-primed fragmentof rodent NMDA receptor subunit cDNA. The preferred human cDNA libraryis a commercially available human hippocampal cDNA library.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other brain regions,may be useful for isolating DNA encoding the human NMDA receptor. Othertypes of libraries include, but are not limited to, cDNA librariesderived from other tissues, cells or cell lines other than humanhippocampal cells, and genomic DNA libraries.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found, for example, in Maniatis, T., Fritsch, E. F., Sambrook,J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press,New York, 2nd edition, 1989).

It is also readily apparent to those skilled in the art that DNAencoding the human NMDA receptor may also be isolated from a suitablegenomic DNA library.

Construction of genomic DNA libraries can be performed by standardtechniques well known in the art. Well known genomic DNA libraryconstruction techiques can be found in Maniatis et al., supra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic comparison of NMDAR1-a, NMDAR1-d, NMDAR1-e.

FIGS. 2A-2E shows the nucleotide sequence (SEQ ID NO:13) of the R1eisoform and a translation of the encoded amino acids (SEQ ID NO:2).

FIGS. 3A-3B depicts the alternative carboxy terminal sequences of thecDNAs encoding the R1a (2613-2854 of SEQ ID NO:11), R1d (2613-2807 ofSEQ ID NO:12) and R1e (2613-2694 of SEQ ID NO:13) isoforms of the humanNMDA R1 receptor subtypes and translations of the encoded amino acids.Deletion 1 (SEQ ID NO:3), deletion 2 (SEQ ID NO:5) and the alternatesequence (SEQ ID NO:7) are also shown with translations of the encodedamino acids.

FIGS. 4A-4J shows the nucleotide sequence (SEQ ID NO:9) of the cDNAencoding the human NMDA R2A receptor subunit and a translation of theencoded amino acids.

FIG. 5 is a schematic representation of cloning vector pMSGneo in which"R" represents the nucleotide sequence of a chosen R1 or R2 subunit ofthe NMDA receptor, and the remainder of the expression vector depictedtherein is derived from the precursor vector pMSGneo and constructed asdescribed in Example 3, steps (a) and (b).

DETAILED DESCRIPTION

Using the preferred method, cDNA clones encoding the human NMDA receptorwere isolated by cDNA library screening. ³² P-radiolabelled fragments ofrodent NMDA receptor NMDA R1 and NMDA R2A subunit cDNAs served as probesfor the isolation of human NMDA receptor cDNA from a commerciallyavailable cDNA library derived from human hippocampal tissue.

For the NMDA R1 subunit, several positively hybridising clones weredetected using the rodent NMDA R1 subunit cDNA probe. The longest ofthese cDNA clones, which was homologous to the published NMDA R1eisoform (Sugihara et al., BBRC, 1992, 185, 826), lacked approximately300 base pairs of the 5' end of the coding region. The missing sequencecould be restored by conventional techniques, which essentially involvedscreening the same hippocampal cDNA library with an EcoRI-SmaI fragmentencoding the last 300 nucleotides of the 5' end of the truncated NMDAR1e clone. A cDNA clone containing the missing sequence could beisolated and engineered by standard methods onto the truncated NMDA R1ecDNA at the internal SmaI site in order to generate a full-length NMDAR1e cDNA, as described in accompanying Example 1.

Analogous techniques applied to other positive clones isolated from thehuman hippocampal cDNA library have yielded cDNAs encoding isoforms ofthe human NMDA R1 receptor subunit corresponding to the published ratNMDA R1a and NMDA R1d isoforms (Sugihara et al., sudra).

For the NMDA R2A subunit, several hybridising clones were detected usingthe rodent NMDA R2A subunit cDNA probe. As with the situationencountered in connection with the R1 subunit as described above, noneof these cDNAs obtained from the human hippocampal cDNA library encodedthe complete human NMDA R2A deduced amino acid sequence. For thisreason, a full-length cDNA was constructed from overlapping truncatedcDNAs, as described in accompanying Example 2.

FIG. 1 depicts the schematic structures of the R1a, R1d and R1e isoformsof the human NMDA R1 receptor subtype. Four putative transmembranesegments (TMI-IV) are represented by solid boxes. R1d has a deletion(deletion II) at the region corresponding to nucleotide residues no.2701-3056 of the R1a cDNA. This deletion results in the generation of anew 22 amino acid carboxyl-terminal (Ct) sequence that follows aminoacid residue 900 of the R1a sequence. R1e has a further deletion(deletion I) and accordingly lacks the sequences of both deletions I andII, thus possessing a structure with the new 22 amino acidcarboxyl-terminal sequence linked to the R1a sequence at position 863.This 22 amino acid carboxyl-terminal sequence, created by alternativesplicing, is indicated in FIG. 1 by hatched boxes.

The sequence for the full-length cDNA encoding the R1e isoform of thehuman NMDA receptor is shown in FIGS. 2A to 2E (SEQ ID NO:13). Thededuced amino acid sequence (SEQ ID NO:2) is shown alongside the cDNAsequence in FIGS. 2A to 2E. The positions of the four putativetransmembrane segments (TM1-4) and of the predicted signal peptide (SP)are indicated in FIGS. 2A to 2E by solid lines.

FIG. 3 depicts the alternative carboxy-terminal sequences of the cDNAsencoding the R1a, R1d and R1e isoforms of the human NMDA R1 receptorsubtype. The nucleotide and deduced amino acid sequences are indicatedin FIG. 3 for each isoform. Indicated amino acid position 863 andnucleotide position 2589 relate to the corresponding positions in thesequence of the R1e isoform depicted in FIGS. 2A to 2E. The nucleotideand deduced amino acid sequences of deletion I (37 amino acids) anddeletion II (38 amino acids) are indicated in FIG. 3. The R1d and R1eisoforms are generated from the R1a isoform by a combination of deletionI and deletions I+II respectively, followed by the addition of thealternative 22 amino acid carboxy-terminal sequence represented by thehatched boxes in FIG. 1, the nucleotide and deduced amino acid sequencesof which are indicated in FIG. 3. The untranslated domains arerepresented in lower case lettering.

The complete nucleotide and deduced amino acid sequences of the cDNAencoding the human NMDA R2A receptor subunit are shown in FIGS. 4A to4J. The cDNA is 4858 bases long. The open reading frame is between bases47 and 4438. Bases 1-46 are 5' untranslated sequence, and bases4439-4858 are 3' untranslated sequence. The deduced amino acid sequenceis 1464 residues long and has 81 differences from the published rat NMDAR2A sequence (Monyer et al., Science, 1992, 256, 1217).

The cloned human NMDA receptor cDNA obtained through the methodsdescribed above may be recombinantly expressed by molecular cloning intoan expression vector containing a suitable promoter and otherappropriate transcription regulatory elements, and then transferring theexpression vector into prokaryotic or eukaryotic host cells to produce arecombinant NMDA receptor. Techniques for such manipulations are fullydescribed in Maniatis et al., supra, and are well known in the art.

DNA encoding the NMDA receptor cloned into an expression vector may thenbe transferred to a recombinant host cell for expression. Recombinanthost cells may be prokaryotic or eukaryotic, including but not limitedto bacteria, yeast, mammalian cells (including but not limited to celllines of human, bovine, porcine, monkey and rodent origin), and insectcells (including but not limited to drosophila derived cell lines). Celllines derived from mammalian species which may be suitable and which arecommercially available include, but are not limited to, L-M (ATCC CCL1), HEK293 (ATCC CCL 1555), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650),COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3(ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCCCCL 26) and MRC-5 (ATCC CCL 171).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, protoplast fusion, and electroporation. The expressionvector-containing cells are clonally propagated and individuallyanalyzed to determine whether they produce NMDA receptor protein.Identification of NMDA receptor expressing host cell clones may be doneby several means, including but not limited to immunological reactivitywith anti-NMDA receptor antibodies, and the presence of hostcell-associated NMDA receptor activity.

Expression of NMDA receptor DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes.

To determine the NMDA receptor cDNA sequence(s) that yield(s) optimallevels of binding activity and/or NMDA receptor protein, NMDA receptorcDNA molecules including but not limited to the following can beconstructed: the full-length open reading frame of the human NMDAreceptor cDNA depicted in FIGS. 2A to 2E or FIGS. 4A to 4J andconstructs containing portions of the cDNA encoding biologically activehuman NMDA receptor protein. All constructs can be designed to containnone, all, or portions of the 5' and 3' untranslated region of humanNMDA receptor cDNA. NMDA receptor activity and levels of proteinexpression can be determined following the introduction, both singly andin combination, of these constructs into appropriate host cells.Following determination of the NMDA receptor cDNA cassette which yieldsoptimal expression, this cDNA construct may be transferred to a varietyof expression host cells, including but not limited to mammalian cells,baculovirus-infected insect cells, E. coli, and the yeast S. cerevisiae.

Expression of the human NMDA receptor in a recombinant host cell affordsNMDA receptor protein in active form. Several purification proceduresare available and suitable for use. Recombinant NMDA receptor maysuitably be purified from cell lysates and extracts, or from conditionedculture medium, by various combinations of, or individual applicationof, salt fractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography.

In addition, recombinant human NMDA receptor can be separated from othercellular proteins by use of an immuno-affinity column made withmonoclonal or polyclonal antibodies specific for the NMDA receptor, orpolypeptide fragments thereof. The preparation and purification ofmonoclonal or polyclonal antibodies specific for the NMDA receptor orpolypeptide fragments thereof can be accomplished by conventionaltechniques well known in the art. Typical procedures include thosedescribed, for example, by Maniatis et al., in Molecular Cloning, ALaboratory manual, Cold Harbor Press, New York, 2nd edition, 1989,Chapter 18.

The present invention is concerned with the production of permanentlytransfected cells containing the NMDA receptor, which will be useful forscreening for drugs which act on this receptor. The NMDA receptor haspreviously been expressed in Xenopus oocytes (Ishii et al., J. Biol.Chem., 1993, 268, 2836) and in transiently transfected mammalian cells(Monyer et al., Science, 1992, 256, 1217). However, both of thosesystems involve transient expression and are unsuitable for screeningpurposes.

We have now achieved the stable expression of the receptor.

Accordingly, the present invention provides a stably co-transfectedeukaryotic cell line capable of expressing an NMDA receptor, whichreceptor comprises at least one R1 subunit isoform, or at least one R1subunit isoform and one or two R2 subunits.

This has been achieved by co-transfecting cells with expression vectors,each harbouring cDNAs encoding for at least one R1 subunit isoform, orfor at least one R1 subunit isoform and one or two R2 subunitsrespectively. In a further aspect, therefore, the present inventionprovides a process for the preparation of a eukaryotic cell line capableof expressing an NMDA receptor, which comprises stably co-transfecting aeukaryotic host cell with one or more expression vectors, at least onesuch vector harbouring the cDNA sequence encoding for an NMDA R1receptor subunit isoform (referred to in the art as the "key subunit"),and optionally other such vectors harbouring the cDNA sequences encodingfor one or two different NMDA R2 receptor subunits. The stable cell linewhich is established expresses an R1 or an R1+R2 NMDA receptorrespectively. Each receptor thereby expressed, comprising a uniquecombination of at least one R1 subunit isoform, or at least one R1subunit isoform and one or two R2 subunits, will be referred tohereinafter as an NMDA receptor "subunit combination". Pharmacologicaland electrophysiological data confirm that the recombinant R1 or R1+R2receptors expressed by the cells of the present invention have theproperties expected of a native NMDA receptor.

As indicated above, expression of the NMDA receptor may be accomplishedby a variety of different promoter-expression systems in a variety ofdifferent host cells. The eukaryotic host cells suitably include yeast,insect and mammalian cells. Preferably the eukaryotic cells which canprovide the host for the expression of the receptor are mammalian cells.Suitable host cells include rodent fibroblast lines, for example mouseLtk⁻, Chinese hamster ovary (CHO) and baby hamster kidney (BHK); HeLa;and HEK293 cells. It is necessary to incorporate at least one R1 subunitisoform, or at least one R1 subunit isoform and one or two R2 subunits,into the cell line in order to produce the required receptor, referredto as R1 or R1+R2 respectively. Within this limitation, the choice ofreceptor subunit combination is made according to the type of activityor selectivity which is being screened for.

In order to employ this invention most effectively for screeningpurposes, it is preferable to build up a library of cell lines, eachwith a different combination of subunits. Typically a library of 11 cellline types is convenient for this purpose. Preferred subunitcombinations include: R1, R1+R2A, R1+R2B, R1+R2C, R1+R2D, R1+R2A+R2B,R1+R2A+R2C, R1+R2A+R2D, R1+R2B+R2C, R1+R2B+R2D and R1+R2C+R2D. Thenomenclature `R1` signifies any one of the seven reported isoforms ofthe NMDA R1 subunit (Sugihara et al., BBRC, 1990, 185, 826): R1a, R1b,R1c, R1d, R1e, R1f and R1g. A particular subunit combination is R1a+R2A.

In a particular embodiment, the present invention provides a stablyco-transfected eukaryotic cell line capable of expressing a human NMDAreceptor comprising the R1a subunit isoform.

In a further embodiment, the present invention provides a stablyco-transfected eukaryotic cell line capable of expressing a human NMDAreceptor comprising the R1a+R2A subunit combination.

As indicated above, the DNAs for the receptor subunits can be obtainedfrom known sources, and are generally obtained as specific nucleotidesequences harboured by a standard cloning vector such as thosedescribed, for example, by Maniatis et al., supra. Preferably the cDNAsequences are derived from the human gene. However, for screeningpurposes, cDNAs from other species are also suitable, such as bovine orrat DNA. Known sources of NMDA receptor subunit cDNAs are as follows:

R1 isoforms (rat): Sugihara et al., BBRC, 1992, 185, 826. Durand et al.,PNAS, 1992, 89, 9359. Hollmann et al., Neuron, 1993, 10, 943.

R2A, R2B, R2C, R2D (rat): Ishii et al., J. Biol. Chem., 1993, 268, 2836.

R2A, R2B, R2C (rat): Monyer et al., Science, 1992, 256, 1217.

R1e (human): Planells-Cases, PNAS, 1993, 90, 5057.

R1a (human): Karp et al., J. Biol. Chem., 1993, 268, 3728.

R1a=(ζ1, R2A=ε1, R2B=ε2, R2C=ε3 (mouse): Kutsuwada et al., Nature, 1992,358, 36.

In another aspect, the invention provides a recombinant expressionvector comprising the nucleotide sequence of an NMDA receptor subunittogether with additional sequences capable of directing the synthesis ofthe said NMDA receptor subunit in cultures of stably co-transfectedeukaryotic cells.

The term "expression vectors" as used herein refers to DNA sequencesthat are required for the transcription of cloned copies of recombinantDNA sequences or genes and the translation of their mRNAs in anappropriate host. Such vectors can be used to express eukaryotic genesin a variety of hosts such as bacteria, blue-green algae, yeast cells,insect cells, plant cells and animal cells. Specifically designedvectors allow the shuttling of DNA between bacteria-yeast,bacteria-plant or bacteria-animal cells. An appropriately constructedexpression vector should contain an origin of replication for autonomousreplication in host cells; selectable markers; a limited number ofuseful restriction enzyme sites; a potential for high copy number; andstrong promoters. A promoter is defined as a DNA sequence that directsRNA polymerase to bind to DNA and to initiate RNA synthesis. A strongpromoter is one which causes mRNAs to be initiated at high frequency.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.

The term "cloning vector" as used herein refers to a DNA molecule,usually a small plasmid or bacteriophage DNA capable of self-replicationin a host organism, and used to introduce a fragment of foreign DNA intoa host cell. The foreign DNA combined with the vector DNA constitutes arecombinant DNA molecule which is derived from recombinant technology.Cloning vectors may include plasmids, bacteriophages, viruses andcosmids.

A variety of mammalian expression vectors may be used to expressrecombinant NMDA receptor in mammalian cells. Commercially availablemammalian expression vectors which may be suitable for recombinant NMDAreceptor expression include, but are not limited to, pCDNAneo(Invitrogen), pCDNAI-Amp (Invitrogen), pCDM8 (Invitrogen), pMSGneo(Proc. Natl. Acad. Sci. USA, 1992, 89, 6378), pMC1neo (Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2)(ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199),pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), andgZD35 (ATCC 37565).

The recombinant expression vector in accordance with the invention maybe prepared by inserting the nucleotide sequence of the chosen NMDAsubunit into a suitable precursor expression vector (hereinafterreferred to as the "precursor vector") using conventional recombinantDNA methodology known from the art. The precursor vector may be obtainedcommercially, or constructed by standard techniques from knownexpression vectors. The precursor vector suitably contains a selectionmarker, typically an antibiotic resistance gene, such as the neomycin orampicillin resistance gene. The precursor vector preferably contains aneomycin resistance gene, adjacent the SV40 early splicing andpolyadenylation region; an ampicillin resistance gene; and an origin ofreplication, e.g. pBR322 ori. The vector also preferably contains aninducible promoter, such as MMTV-LTR (inducible with dexamethasone) ormetallothionin (inducible with zinc), so that transcription can becontrolled in the cell line of this invention. This reduces or avoidsany problem of toxicity in the cells because of the ion channelintrinsic to the NMDA receptor.

One suitable precursor vector is pMAMneo, available from ClontechLaboratories Inc. (Lee et al., Nature, 1981, 294, 228; and Sardet etal., Cell, 1989, 56, 271). Alternatively the precursor vector pMSGneocan be constructed from the vectors pMSG and pSV2neo as described inExample 3 herein.

The recombinant expression vector of the present invention is thenproduced by cloning the NMDA receptor subunit cDNA into the aboveprecursor vector. The required receptor subunit cDNA is subcloned fromthe vector in which it is harboured, and ligated into a restrictionenzyme site in the polylinker of the precursor vector, for examplepMAMneo or pMSGneo, by standard cloning methodology known from the art,and in particular by techniques analogous to those described in Example3, step (b) herein.

One suitable expression vector of the present invention is illustratedin FIG. 5 of the accompanying drawings, in which R represents thenucleotide sequence of a chosen R1 or R2 subunit of the NMDA receptor,and the remainder of the expression vector depicted therein is derivedfrom the precursor vector pMSGneo and constructed as described inaccompanying Example 3, steps (a) and (b).

For each cell line of the present invention, one or more such vectorswill be necessary. At least one such vector will contain the cDNAsequence encoding for an R1 subunit isoform. Vectors containing the cDNAsequences encoding for one or two different R2 subunits may also beutilised.

Cells are then co-transfected with the desired combination of one ormore, typically one, two or three, expression vectors. There are severalcommonly used techniques for transfection of eukaryotic cells in vitro.Calcium phosphate precipitation of DNA is most commonly used (Bachettiet al., Proc. Natl. Acad. Sci. USA, 1977, 74, 1590-1594; Maitland etal., Cell, 1977, 14, 133-141), and represents a favoured technique inthe context of the present invention.

A small percentage of the host cells takes up the recombinant DNA. In asmall percentage of those, the DNA will integrate into the host cellchromosome. Because the neomycin resistance gene will have beenincorporated into these host cells, they can be selected by isolatingthe individual clones which will grow in the presence of neomycin. Eachsuch clone is then tested to identify those which will produce thereceptor. This is achieved by inducing the production, for example withdexamethasone, and then detecting the presence of receptor by means ofradioligand binding.

In a further aspect, the present invention provides protein preparationsof NMDA receptor subunit combinations, especially human NMDA receptorsubunit combinations, derived from cultures of stably transfectedeukaryotic cells. The invention also provides preparations of membranescontaining subunit combinations of the NMDA receptor, especially humanNMDA receptor subunit combinations, derived from cultures of stablytransfected eukaryotic cells. In particular, the protein preparationsand membrane preparations according to the invention will suitablycontain the R1, R1+R2A, R1+R2B, R1+R2C, R1+R2D, R1+R2A+R2B, R1+R2A+R2C,R1+R2A+R2D, R1+R2B+R2C, R1+R2B+R2D or R1+R2C+R2D subunit combinations ofthe human NMDA receptor, wherein the nomenclature `R1` signifies any oneof the several isoforms of the R1 subunit as described above. Apreferred subunit combination is the R1a+R2A subunit combination. In anespecially preferred embodiment, the invention provides cell membranescontaining a human NMDA receptor consisting of the R1a+R2A subunitcombination isolated from stably transfected mouse Ltk⁻ fibroblastcells.

The cell line, and the membrane preparations therefrom, according to thepresent invention have utility in screening and design of drugs whichact upon the NMDA receptor. The present invention accordingly providesthe use of the cell line described above, of membrane preparationsderived therefrom, and of the cloned human NMDA receptor as describedherein, in screening for and designing medicaments which interactselectively with the NMDA receptor. Of particular interest in thiscontext are molecules capable of interacting selectively with NMDAreceptors made up of varying subunit combinations. As will be readilyapparent, the cell line in accordance with the present invention, andthe membrane preparations derived therefrom, provide ideal systems forthe study of structure, pharmacology and function of the various NMDAreceptor subtypes.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an MRNA moleculewhich encodes the human NMDA receptor, so as to prevent translation ofthe mRNA molecule. The antisense oligonucleotide may have a sequencecapable of binding specifically with any sequences of the cDNA moleculewhose sequence is shown in, for example, FIG. 3 or FIGS. 4A-4J. Aparticular example of an antisense oligonucleotide is an antisenseoligonucleotide comprising chemical analogues of nucleotides.

This invention provides a transgenic nonhuman mammal expressing DNAencoding the human NMDA receptor. This invention also provides atransgenic nonhuman mammal, so mutated as to be incapable of normalreceptor activity, and not expressing the native NMDA receptor, whichmammal is nonetheless capable of expressing DNA encoding the human NMDAreceptor. This invention further provides a transgenic nonhuman mammalwhose genome comprises antisense DNA complementary to DNA encoding thenative NMDA receptor, so placed as to be transcribed into antisense mRNAwhich is complementary to mRNA encoding the receptor and whichhybridizes to MRNA encoding the receptor thereby reducing itstranslation. The DNA may additionally comprise an inducible promoter oradditionally comprise tissue specific regulatory elements, so thatexpression can be induced, or restricted to specific cell types.Examples of DNA are DNA or cDNA molecules having a coding sequencesubstantially the same as the coding sequence shown in, for example,FIG. 3 or FIGS. 4A to 4J. An example of a transgenic animal is atransgenic mouse. Examples of tissue specificity-determining regions arethe metallothionein promoter (Low et al., Science, 1986, 231, 1002-1004)and the L7 promoter (Oberdick et al., Science, 1990, 248, 223-226).

The following non-limiting Examples illustrate the present invention.

EXAMPLE 1 Isolation of cDNAs Encoding Three Different Isoforms of theHuman NMDA R1 Receptor Subunit

1. Methods

Oligonucleotide primers derived from the published rat NMDA R1 receptorsequence (Moriyoshi et al., Nature (London), 1991, 354, 31-37) [0₁ : 5'GAC/CCC/AGG/CTC/AGA/ATT/CCC/TCA/GAC/AAG3' SEQ ID NO:14 and 30₂ : 5'CAC/CAG/GAA/GAA/GTC/TGC/CAT/GTT/CTC/A3' SEQ ID NO:15 ] were used in thepolymerase chain reaction (PCR) to isolate a 420 bp cDNA encoding theputative large cytoplasmic loop of the human NMDA R1 receptor subtype. Ahuman hippocampal cDNA library was screened at low stringency (55° C. in5×SSPE, 5×Denhardt's solution, 0.1% SDS, 100 μg/ml salmon sperm DNA)using this partial PCR cDNA as a [³² P]-labelled probe containing0.5×10⁶ cpm/ml. Twenty positive clones encoding putative NMDA receptorswere isolated. The longest clone, homologous to the published NMDA R1e,0.6 kb long, was lacking approximately 300 bp of the 5' end of thecoding region. This missing sequence was obtained by screening the samehuman hippocampal cDNA library with an EcoR1-SmaI fragment encoding thelast 300 nucleotides of the 5' end of the truncated NMDA R1e clone. AcDNA clone containing the missing sequence was isolated and engineeredonto the truncated NMDA R1e cDNA at the internal SmaI site in order togenerate a full length NMDA R1e cDNA. This full length human NMDA R1eclone contains a 2.8 kb coding sequence flanked by 0.05 kb and 0.6 kb ofthe 5' and the 3' untranslated regions respectively.

2. Results

The sequence of the human NMDA R1e cDNA (FIG. 2A to 2E) is homologous(98.6% of homology) with the published rat NMDA R1e cDNA (Sugihara etal., BBRC, 1992, 185, 826-832). The amino acid sequence of theC-terminal domain of this NMDA R1e subunit differs from the originalpublished rat NMDA R1a subunit (Moriyoshi et al., Nature (London), 1991,354, 31-37). Among the twenty positive clones isolated from a humanhippocampal cDNA library, screened with the 420 bp long PCR fragment,two other clones have revealed a sequence homologous to the NMDA R1e;they are the human version of the published rat NMDA R1a and NMDA R1d,respectively (Sugihara et al., BBRC, 1992, 185, 826-832). FIG. 1 showsthe schematic structures of the three human NMDA R1a, R1d and R1eisoforms, on the basis of the structural comparison with the originalNMDA R1 (now referred to as R1a). R1d has a deletion (deletion II, whosenucleotide and deduced amino acid sequences are shown in FIG. 3) at theregion corresponding to nucleotide residues no. 2701-3056 of the R1acDNA. This deletion results in the generation of a new 22 amino acidcarboxyl-terminal sequence that follows amino acid residue 900 of theR1a sequence. The nucleotide and deduced amino acid sequences of thisnew 22 amino acid sequence are depicted in FIG. 3. R1e has a furtherdeletion (deletion I, whose nucleotide and deduced amino acid sequencesare shown in FIG. 3) and accordingly lacks the sequences of bothdeletions I and II, thus possessing a structure with the new 22 aminoacid carboxyl-terminal sequence (cf. FIG. 3) linked to the R1a sequenceat position 863.

EXAMPLE 2 Isolation of cDNAs Encoding the Human NMDA R2A Subunit

Oligonucleotide primers derived from the published rat NMDA R2A cDNAsequence (Monyer et al., Science, 1992, 256, 1217) were used in thepolymerase chain reaction (PCR) to generate N-terminal and C-terminalcDNA probes. Primers for N-terminal probe (encoding residuesLeu16-Met74) were (SEQ ID NO:16) 5'TGCCGGGAATTCTGGTCTGGCGCGATCCGG3' and(SEQ ID NO:17) 5'GGGTCAAGCTTTTCATCAATAACGCCAC3'. Primers for theC-terminal probe (encoding residues Leu898-Gln1209) were SEQ ID NO:185'TGCTAAGATTCTTCGGTCAGCTAAAAAC3' SEQ ID NO:19 and5'AATGTGAAGCTTTCTGCCGGTATCGATCAC3'. The sense oligonucleotide (the firstof each pair) had an EcoRI restriction enzyme site incorporated into it,while the antisense oligonucleotide had a HindIII restriction enzymesite incorporated into it. PCR using rat brain cDNA as template wasperformed using standard techniques (e.g. Whiting et al., Proc. Natl.Acad. Sci. USA, 1990, 87, 9966-9970) to yield PCR products of 185 and950 bases in length (N-terminal cDNA probe and C-terminal cDNA probe,respectively). These were subcloned into pBluescript Sk⁻ (Stratagene). Ahuman hippocampal cDNA library (Stratagene) was screened under moderatestringency conditions (42° C. in 5×SSPE, 5×Denhardt's solution, 100mg/ml salmon sperm DNA, 0.1% sodium dodecyl sulphate, 30% formamide)using standard techniques (e.g. Maniatis et al., in Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, New York, 2nd edition,1989). A number of cDNA clones were isolated and characterised. ThesecDNAs encoded the entire human NMDA R2A receptor subunit, with theexception of a sequence spanning bases approx. 300-900 of the codingregion. This missing sequence was obtained by PCR: oligonucleotideprimers derived from the 5' untranslated region of the human NMDA R2Areceptor subunit cDNA sequence SEQ ID NO:20(5'CCCCACTGCATCCGGTACCTTCTCGGGCTACA3') and the coding region of thehuman NMDA R2A receptor subunit cDNA sequence, FIGS. 4A to 4J, bases873-902 SEQ ID NO:21 (5'AGTCCCAGTCATATGAGACAGAAATGA3'), were used togenerate a 5' cDNA fragment using human hippocampal cDNA as template. Afull-length human NMDA R2A receptor subunit cDNA was generated byassembling overlapping cDNAs using standard molecular biologytechniques, and the 5' cDNA fragment generated by PCR and sequenced onboth strands using Taq dideoxy terminator cycle sequencing (AppliedBioSystems) and an Applied BioSystems 373A DNA Sequencer. The completenucleotide and deduced amino acid sequence is shown in FIGS. 4A to 4J(SEQ ID NO:9 and 10)

EXAMPLE 3 Preparation of R1a transfected Cells and R1a+R2A TransfectedCells

a) Construction of Eukaryotic Expression Vector pMSGneo

The approx. 2500 base pair HindIII-EcoRI fragment of the vector pMSG(purchased from Pharmacia Biosystems Limited, Milton Keynes, UnitedKingdom), containing the gpt structural gene and SV40 polyadenylationsignals was replaced by the approx. 2800 base pair HindIII-EcoRIfragment of pSV2neo (Southern, P. J. and Berg, P. J., Molecular andApplied Genetics, 1, 327-341, 1982) containing the neomycin resistancegene Neo^(r) and SV40 polyadenylation signals. The EcoRI and HindIIIsites were then removed by restriction digesting, blunt ending withklenow polymerase, and religating. EcoRI and HindIII cloning sites werethen inserted at the XhoI and SmaI sites of the polylinker byconventional techniques using EcoRI and HindIII linkers.

b) Cloning of Subunit cDNAs into pMSGneo

Human R1a and R2A cDNA were cloned as described in Examples 1 and 2above respectively. Human R1a was subcloned from pBluescript Sk⁻(Stratagene, San Diego, Calif., USA) by digestion with SalI and EcoRIand ligation into the SalI and EcoRI sites of pMSGneo. Human R2A wassubcloned from pBluescript Sk⁻ by restriction digestion with NheI andXhoI and ligation into the NheI and XhoI sites of pMSGneo.

c) Co-transfection of Mouse Ltk⁻ Cells

Ltk⁻ cells were obtained from the Salk Institute for Biological Studies,San Diego, Calif. Cells were grown at 37° C., 5-8% CO₂, in ModifiedEagles Medium containing penicillin, streptomycin and 10% fetal calfserum. The expression vector harbouring the NMDA receptor subunit DNAsfor co-transfection was prepared by a standard protocol (Chen, C. andOkayama, H., BioTechniques, 6, 632-638, 1988). For co-transfection, Ltk⁻cells were plated in dishes (approx. 2×10⁵ cells/dish) and grownovernight. The transfection was performed by calcium phosphateprecipitation using a kit (purchased from 5 Prime→3 Prime Products,Westchester, Pa.). Co-transfection was performed according tomanufacturers' instructions, using 5 μg of each subunit DNA constructper 10 cm dish of cells. After 2 days in culture the cells were divided1:8 into culture medium containing 1 mg/ml neomycin [Geneticin(obtainable from Gibco BRL, Paisley, Scotland, United Kingdom)]. After afurther week the concentration was increased to 1.5 mg/ml, and then 2mg/ml 1 week after that. Resistant clones of cells were isolated andsubcloned using cloning cylinders. Subclones were analysed usingradioligand binding: subclones were grown in 10 cm culture dishes, andwhen confluent changed into culture medium containing 1 μM dexamethasone(obtainable from Sigma Chemical Company, Poole, Dorset, United Kingdom).In order to prevent cell death induced by the expression of therecombinant R1a+R2A receptor, 10 mM MgCl₂ and 100 μM D-AP5(D-2-amino-5-phosphonovalerate) (Tocris-Neuramin, Essex, United Kingdom)were added into the culture medium in addition to the dexamethasone.MgCl₂ and D-AP5 are substances known to act as antagonists at the NMDAreceptor (Wong and Kemp, Ann. Rev. Pharmacol. Toxicol., 1991, 31, 401).3-5 days later the cells were harvested, membranes prepared and used forradioligand binding (see Example 4, step (a) below), using the glycinesite antagonist [³ H]-L-689,560 (custom synthesised by AmershamInternational p1c, Amersham, United Kingdom) for the characterisation ofthe recombinant R1 receptor, and the channel blocker [³ H]-dizocilpine(obtained from New England Nuclear, Du Pont Ltd., Stevenage, UnitedKingdom) for the characterisation of the recombinant R1a+R2A receptor.

The recombinant R1a receptor clone expressing the highest amount of [³H]-L-689,560 binding was subcloned from a single cell by limitingdilution. The resultant clonal population of these cells is referred tohereinbelow as population A.

The recombinant R1a+R2A receptor clone expressing the highest amount of[³ H]-dizocilpine binding was subcloned from a single cell by limitingdilution. The resultant clonal population of these cells is referred tohereinbelow as population B.

EXAMPLE 4 Characterization of R1a Transfected Cells and R1a+R2ATransfected Cells

a) Radioligand Binding

The nature of the recombinant R1a NMDA receptors prepared as describedin Example 3 was addressed by characterization of the glycine sitebinding pharmacology, using the glycine site antagonist [³ H]-L-689,560.For radioligand binding assays, cells which had been induced by culturein dexamethasone containing medium for 3-5 days were scraped off into 5mM Tris-Acetate, pH 7.0 at 4° C. (buffer 1) and pelleted (20,000 rpm,Sorvall RC5C centrifuge). The cell pellet was resuspended in buffer 1,homogenised using an Ultra-Turrax homogeniser and then pelleted asabove. This was repeated once more, and the cells then resuspended in 50mM Tris-Acetate, pH 7.0 at 4° C. (buffer 2) at a protein concentrationof 1 mg/ml. Radioligand binding was performed in 0.5 ml final volumebuffer 2, containing 1 nM of [³ H]-L-689,560 and 100 μg of protein.After 2 hours incubation on ice the membranes were harvested ontofilters using a Brandel cell harvester, washed with cold buffer 2, andbound radioactivity determined by scintillation counting. Non-specificbinding was determined in a parallel incubation containing 10 mMglycine. The recombinant R1a receptors bound [³ H]-L-689,560 at levelsof up to 3700 fmols/mg protein. No binding was seen to untransfectedLtk⁻ cells, confirming that the [³ H]-L-689,560 was binding torecombinant R1 NMDA receptors.

The nature of the recombinant R1a+R2A NMDA receptors prepared asdescribed in Example 3 was addressed by demonstrating binding of thechannel blocker [³ H]-dizocilpine. For radioligand binding assays, cellswhich had been induced by culture in dexamethasone, MgCl₂ and D-AP5containing medium for 3-5 days were scraped off into 5 mM Tris-Acetate,pH 7.0 at 4° C. (buffer 1) and pelleted (20,000 rpm, Sorvall RC5Ccentrifuge). The cell pellet was resuspended in buffer 1, homogenisedusing an Ultra-Turrax homogeniser and then pelleted as above. This wasrepeated once more, and the cells then resuspended in 5 mM Tris-Acetate,pH 7.4 at 25° C. (buffer 3) at a protein concentration of 1 mg/ml.Radioligand binding was performed in 0.5 ml final volume buffer 3,containing 2 nM of [³ H]-dizocilpine and 100 μg of protein. After 2hours at 25° C. in the presence of 30 μM glycine and 30 μM L-glutamatethe membranes were washed with buffer 1 and bound radioactivitydetermined by scintillation counting. The recombinant R1a+R2A receptorsbound [³ H]-dizocilpine at levels up to 200 fmols/mg protein. No bindingwas seen to untransfected Ltk⁻ cells, confirming that the [³H]-dizocilpine was binding to recombinant R1a+R2A NMDA receptors.Non-specific binding was determined in a parallel incubation containing100 μM dizocilpine. The recombinant R1a+R2A receptors bound also [³H]-L-689,560 at levels of up to 500 fmols/mg protein, confirming thepresence of a glycine binding site in population B cells.

b) Electrophysiology

The nature of the NMDA receptor expressed by population B cells has beenextensively characterised by electrophysiological techniques, usingwhole cell patch clamp. Only cells induced by culture in the presence ofdexamethasone showed responses to NMDA. Concentration response curves toglutamate in a saturating concentration of glycine (10 μM) gave a logEC₅₀ of 5.77, and a Hill coefficient of 1.1. The response to glutamateand glycine was antagonised by the competitive glutamate receptorantagonist CGS 19,755 (Research Biochemical International, MA, USA). Allthese electrophysiological data confirm that the recombinant R1a+R2ANMDA receptor expressed by population B cells has the propertiesexpected of a bona fide NMDA receptor.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 21                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2621 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GCCGCGCAGA GCCAGGCCCG CGGCCCGAGC CCATGAGCAC CATGCGCCTG TT - #GACGCTCG         60                                                                          - CCCTGCTGTT CTCCTGCTCC CTCGCCCGTG CCGCGTGCGA CCCCAAGATC GT - #CAACATTG        120                                                                          - GCGCGGTGCT GAGCACGCGG AAGCACGAGC AGATGTTCCG CGAGGCCGTG AA - #CCAGGCCA        180                                                                          - ACAAGCGGCA CGGCTCCTGG AAGATTCAGC TCAATGCCAC CTCCGTCACG CA - #CAAGCCCA        240                                                                          - ACGCCATCCA GATGGCTCTG TCGGTGTGCG AGGACCTCAT CTCCAGCCAG GT - #CTACGCCA        300                                                                          - TCCTAGTTAG CCATCCACCT ACCCCCAACG ACCACTTCAC TCCCACCCCT GT - #CTCCTACA        360                                                                          - CAGCCGGCTT CTACCGCATA CCCGTGCTGG GGCTGACCAC CCGCATGTCC AT - #CTACTCGG        420                                                                          - ACAAGAGCAT CCACCTGAGC TTCCTGCGCA CCGTGCCGCC CTACTCCCAC CA - #GTCCAGCG        480                                                                          - TGTGGTTTGA GATGATGCGT GTCTACAGCT GGAACCACAT CATCCTGCTG GT - #CAGCGACG        540                                                                          - ACCACGAGGG CCGGGCGGCT CAGAAACGCC TGGAGACGCT GCTGGAGGAG CG - #TGAGTCCA        600                                                                          - AGGCAGAGAA GGTGCTGCAG TTTGACCCAG GGACCAAGAA CGTGACGGCC CT - #GCTGATGG        660                                                                          - AGGCGAAAGA GCTGGAGGCC CGGGTCATCA TCCTTTCTGC CAGCGAGGAC GA - #TGCTGCCA        720                                                                          - CTGTATACCG CGCAGCCGCG ATGCTGAACA TGACGGGCTC CGGGTACGTG TG - #GCTGGTCG        780                                                                          - GCGAGCGCGA GATCTCGGGG AACGCCCTGG CCTACGCCCC AGACGGCATC CT - #CGGGCTGC        840                                                                          - AGCTCATCAA CGGCAAGAAC GAGTCGGCCC ACATCAGCGA CGCCGTGGGC GT - #GGTGGCCC        900                                                                          - AGGCCGTGCA CGAGCTCCTC GAGAAGGAGA ACATCACCGA CCCGCCGCGG GG - #CTGCGTGG        960                                                                          - GCAACACCAA CATCTGGAAG ACCGGGCCGC TCTTCAAGAG AGTGCTGATG TC - #TTCCAAGT       1020                                                                          - ATGCGGATGG GGTGACTGGT CGCGTGGAGT TCAATGAGGA TGGGGACCGG AA - #GTTCGCCA       1080                                                                          - ACTACAGCAT CATGAACCTG CAGAACCGCA AGCTGGTGCA AGTGGGCATC TA - #CAATGGCA       1140                                                                          - CCCACGTCAT CCCTAATGAC AGGAAGATCA TCTGGCCAGG CGGAGAGACA GA - #GAAGCCTC       1200                                                                          - GAGGGTACCA GATGTCCACC AGACTGAAGA TTGTGACGAT CCACCAGGAG CC - #CTTCGTGT       1260                                                                          - ACGTCAAGCC CACGCTGAGT GATGGGACAT GCAAGGAGGA GTTCACAGTC AA - #CGGCGACC       1320                                                                          - CAGTCAAGAA GGTGATCTGC ACCGGGCCCA ACGACACGTC GCCGGGCAGC CC - #CCGCCACA       1380                                                                          - CGGTGCCTCA GTGTTGCTAC GGCTTTTGCA TCGACCTGCT CATCAAGCTG GC - #ACGGACCA       1440                                                                          - TGAACTTCAC CTACGAGGTG CACCTGGTGG CAGATGGCAA GTTCGGCACA CA - #GGAGCGGG       1500                                                                          - TGAACAACAG CAACAAGAAG GAGTGGAATG GGATGATGGG CGAGCTGCTC AG - #CGGGCAGG       1560                                                                          - CAGACATGAT CGTGGCGCCG CTAACCATAA ACAACGAGCG CGCGCAGTAC AT - #CGAGTTTT       1620                                                                          - CCAAGCCCTT CAAGTACCAG GGCCTGACTA TTCTGGTCAA GAAGGAGATT CC - #CCGGAGCA       1680                                                                          - CGCTGGACTC GTTCATGCAG CCGTTCCAGA GCACACTGTG GCTGCTGGTG GG - #GCTGTCGG       1740                                                                          - TGCACGTGGT GGCCGTGATG CTGTACCTGC TGGACCGCTT CAGCCCCTTC GG - #CCGGTTCA       1800                                                                          - AGGTGAACAG CGAGGAGGAG GAGGAGGACG CACTGACCCT GTCCTCGGCC AT - #GTGGTTCT       1860                                                                          - CCTGGGGCGT CCTGCTCAAC TCCGGCATCG GGGAAGGCGC CCCCAGAAGC TT - #CTCAGCGC       1920                                                                          - GCATCCTGGG CATGGTGTGG GCCGGCTTTG CCATGATCAT CGTGGCCTCC TA - #CACTGCCA       1980                                                                          - ACTTGGCGGC CTTCCTGGTG CTGGACCGGC CGGAGGAGCG CATCACGGGC AT - #CAACGACC       2040                                                                          - CTCGGCTGAG GAACCCCTCG GACAAGTTTA TCTACGCCAC GGTGAAGCAG AG - #CTCCGTGG       2100                                                                          - ATATCTACTT CCGGCGCCAG GTGGAGCTGA GCACCATGTA CCGGCATATG GA - #GAAGCACA       2160                                                                          - ACTACGAGAG TGCGGCGGAG GCCATCCAGG CCGTGAGAGA CAACAAGCTG CA - #TGCCTTCA       2220                                                                          - TCTGGGACTC GGCGGTGCTG GAGTTCGAGG CCTCGCAGAA GTGCGACCTG GT - #GACGACTG       2280                                                                          - GAGAGCTGTT TTTCCGCTCG GGCTTCGGCA TAGGCATGCG CAAAGACAGC CC - #CTGGAAGC       2340                                                                          - AGAACGTCTC CCTGTCCATC CTCAAGTCCC ACGAGAATGG CTTCATGGAA GA - #CCTGGACA       2400                                                                          - AGACGTGGGT TCGGTATCAG GAATGTGACT CGCGCAGCAA CGCCCCTGCA AC - #CCTTACTT       2460                                                                          - TTGAGAACAT GGCCGGGGTC TTCATGCTGG TAGCTGGGGG CATCGTGGCC GG - #GATCTTCC       2520                                                                          - TGATTTTCAT CGAGATTGCC TACAAGCGGC ACAAGGATGC TCGCCGGAAG CA - #GATGCAGC       2580                                                                          # 2621             TAAC GTGTGGCGGA AGAACCTGCA G                               - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 863 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Ser Thr Met Arg Leu Leu Thr Leu Ala Le - #u Leu Phe Ser Cys Ser         #                15                                                           - Leu Ala Arg Ala Ala Cys Asp Pro Lys Ile Va - #l Asn Ile Gly Ala Val         #            30                                                               - Leu Ser Thr Arg Lys His Glu Gln Met Phe Ar - #g Glu Ala Val Asn Gln         #        45                                                                   - Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gl - #n Leu Asn Ala Thr Ser         #    60                                                                       - Val Thr His Lys Pro Asn Ala Ile Gln Met Al - #a Leu Ser Val Cys Glu         #80                                                                           - Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Le - #u Val Ser His Pro Pro         #                95                                                           - Thr Pro Asn Asp His Phe Thr Pro Thr Pro Va - #l Ser Tyr Thr Ala Gly         #           110                                                               - Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Th - #r Arg Met Ser Ile Tyr         #       125                                                                   - Ser Asp Lys Ser Ile His Leu Ser Phe Leu Ar - #g Thr Val Pro Pro Tyr         #   140                                                                       - Ser His Gln Ser Ser Val Trp Phe Glu Met Me - #t Arg Val Tyr Ser Trp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn His Ile Ile Leu Leu Val Ser Asp Asp Hi - #s Glu Gly Arg Ala Ala         #               175                                                           - Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Ar - #g Glu Ser Lys Ala Glu         #           190                                                               - Lys Val Leu Gln Phe Asp Pro Gly Thr Lys As - #n Val Thr Ala Leu Leu         #       205                                                                   - Met Glu Ala Lys Glu Leu Glu Ala Arg Val Il - #e Ile Leu Ser Ala Ser         #   220                                                                       - Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Al - #a Ala Met Leu Asn Met         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Gl - #u Arg Glu Ile Ser Gly         #               255                                                           - Asn Ala Leu Ala Tyr Ala Pro Asp Gly Ile Le - #u Gly Leu Gln Leu Ile         #           270                                                               - Asn Gly Lys Asn Glu Ser Ala His Ile Ser As - #p Ala Val Gly Val Val         #       285                                                                   - Ala Gln Ala Val His Glu Leu Leu Glu Lys Gl - #u Asn Ile Thr Asp Pro         #   300                                                                       - Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Tr - #p Lys Thr Gly Pro Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Al - #a Asp Gly Val Thr Gly         #               335                                                           - Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Ly - #s Phe Ala Asn Tyr Ser         #           350                                                               - Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gl - #n Val Gly Ile Tyr Asn         #       365                                                                   - Gly Thr His Val Ile Pro Asn Asp Arg Lys Il - #e Ile Trp Pro Gly Gly         #   380                                                                       - Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Se - #r Thr Arg Leu Lys Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Thr Ile His Gln Glu Pro Phe Val Tyr Va - #l Lys Pro Thr Leu Ser         #               415                                                           - Asp Gly Thr Cys Lys Glu Glu Phe Thr Val As - #n Gly Asp Pro Val Lys         #           430                                                               - Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Se - #r Pro Gly Ser Pro Arg         #       445                                                                   - His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cy - #s Ile Asp Leu Leu Ile         #   460                                                                       - Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Gl - #u Val His Leu Val Ala         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asp Gly Lys Phe Gly Thr Gln Glu Arg Val As - #n Asn Ser Asn Lys Lys         #               495                                                           - Glu Trp Asn Gly Met Met Gly Glu Leu Leu Se - #r Gly Gln Ala Asp Met         #           510                                                               - Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Ar - #g Ala Gln Tyr Ile Glu         #       525                                                                   - Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Th - #r Ile Leu Val Lys Lys         #   540                                                                       - Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Me - #t Gln Pro Phe Gln Ser         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Thr Leu Trp Leu Leu Val Gly Leu Ser Val Hi - #s Val Val Ala Val Met         #               575                                                           - Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gl - #y Arg Phe Lys Val Asn         #           590                                                               - Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Le - #u Ser Ser Ala Met Trp         #       605                                                                   - Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Il - #e Gly Glu Gly Ala Pro         #   620                                                                       - Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Va - #l Trp Ala Gly Phe Ala         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Le - #u Ala Ala Phe Leu Val         #               655                                                           - Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Il - #e Asn Asp Pro Arg Leu         #           670                                                               - Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Th - #r Val Lys Gln Ser Ser         #       685                                                                   - Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Le - #u Ser Thr Met Tyr Arg         #   700                                                                       - His Met Glu Lys His Asn Tyr Glu Ser Ala Al - #a Glu Ala Ile Gln Ala         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Val Arg Asp Asn Lys Leu His Ala Phe Ile Tr - #p Asp Ser Ala Val Leu         #               735                                                           - Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Va - #l Thr Thr Gly Glu Leu         #           750                                                               - Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Ar - #g Lys Asp Ser Pro Trp         #       765                                                                   - Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Se - #r His Glu Asn Gly Phe         #   780                                                                       - Met Glu Asp Leu Asp Lys Thr Trp Val Arg Ty - #r Gln Glu Cys Asp Ser         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Gl - #u Asn Met Ala Gly Val         #               815                                                           - Phe Met Leu Val Ala Gly Gly Ile Val Ala Gl - #y Ile Phe Leu Ile Phe         #           830                                                               - Ile Glu Ile Ala Tyr Lys Arg His Lys Asp Al - #a Arg Arg Lys Gln Met         #       845                                                                   - Gln Leu Ala Phe Ala Ala Val Asn Val Trp Ar - #g Lys Asn Leu Gln             #   860                                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 111 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - GATAGAAAGA GTGGTAGAGC AGAGCCTGAC CCTAAAAAGA AAGCCACATT TA - #GGGCTATC         60                                                                          #            111CTTCCAG CTTCAAGAGG CGTAGGTCCT CCAAAGACAC G                    - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 37 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Asp Arg Lys Ser Gly Arg Ala Glu Pro Asp Pr - #o Lys Lys Lys Ala Thr         #                15                                                           - Phe Arg Ala Ile Thr Ser Thr Leu Ala Ser Se - #r Phe Lys Arg Arg Arg         #            30                                                               - Ser Ser Lys Asp Thr                                                                 35                                                                    - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 184 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - AGCACCGGGG GTGGACGCGG CGCTTTGCAA AACCAAAAAG ACACAGTGCT GC - #CGCGACGG         60                                                                          - CCTATTGAGA GGGAGGAGGG CCAGCTGCAG CTGTGTTCCC GTCATAGGGA GA - #GCTGAGAC        120                                                                          - TCCCCGCCCG CCCTCCTCTG CCCCCCTCCC CCGCAGACAG ACAGACAGAC GG - #ACGGGACA        180                                                                          #            184                                                              - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 38 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Ser Thr Gly Gly Gly Arg Gly Ala Leu Gln As - #n Gln Lys Asp Thr Val         #                15                                                           - Leu Pro Arg Arg Pro Ile Glu Arg Glu Glu Gl - #y Gln Leu Gln Leu Cys         #            30                                                               - Ser Arg His Arg Glu Ser                                                             35                                                                    - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 103 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - CAGTACCATC CCACTGATAT CACGGGCCCG CTCAACCTCT CAGATCCCTC GG - #TCAGCACC         60                                                                          #103               GGAG CGCCCACCTG CCCAGTTTAG CCC                             - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Gln Tyr His Pro Thr Asp Ile Thr Gly Pro Le - #u Asn Leu Ser Asp Pro         #                15                                                           - Ser Val Ser Thr Val Val                                                                 20                                                                - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 4858 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - CTGCATCCTC GACCTTCTCG GGCTACAGGG ACCGTCAGTG GCGACTATGG GC - #AGAGTGGG         60                                                                          - CTATTGGACC CTGCTGGTGC TGCCGGCCCT TCTGGTCTGG CGCGGTCCGG CG - #CCGAGCGC        120                                                                          - GGCGGCGGAG AAGGGTCCCC CCGCGCTAAA TATTGCGGTG ATGCTGGGTC AC - #AGCCACGA        180                                                                          - CGTGACAGAG CGCGAACTTC GAACACTGTG GGGCCCCGAG CAGGCGGGCG GG - #CTGCCCCT        240                                                                          - GGACGTGAAC GTGGTAGCTC TGCTGATGAA CCGCACCGAC CCCAAGAGCC TC - #ATCACGCA        300                                                                          - CGTGTGCGAC CTCATGTCCG GGGCACGCAT CCACGGCCTC GTGTTTGGGG AC - #GACACGGA        360                                                                          - CCAGGAGGTC GTAGCCCAGA TGCTGGATTT TATCTCCTCC CACACCTTCG TC - #CCCATCTT        420                                                                          - GGGCATTCAT GGGGGCGCAT CTATGATCAT GGCTGACAAG GATCCGACGT CT - #ACCTTCTT        480                                                                          - CCAGTTTGGA GCGTCCATCC AGCAGCAAGC CACGGTCATG CTGAAGATCA TG - #CAGGATTA        540                                                                          - TGACTGGCAT GTCTTCTCCC TGGTGACCAC TATCTTCCCT GGCTACAGGG AA - #TTCATCAG        600                                                                          - CTTCGTCAAG ACCACAGTGG ACAACAGCTT TGTGGGCTGG GACATGCAGA AT - #GTGATCAC        660                                                                          - ACTGGACACT TCCTTTGAGG ATGCAAAGAC ACAAGTCCAG CTGAAGAAGA TC - #CACTCTTC        720                                                                          - TGTCATCTTG CTCTACTGTT CCAAAGACGA GGCTGTTCTC ATTCTGAGTG AG - #GCCCGCTC        780                                                                          - CCTTGGCGTC ACCGGGTATG ATTTCTTCTG GATTGTCCCC AGCTTGGTCT CT - #GGGAACAC        840                                                                          - GGAGCTCATC CCAAAAGAGT TTCCATCGGG ACTCATTTCT GTCTCCTACG AT - #GACTGGGA        900                                                                          - CTACAGCCTG GAGGCGAGAG TGAGGGACGG CATTGGCATC CTAACCACCG CT - #GCATCTTC        960                                                                          - TATGCTGGAG AAGTTCTCCT ACATCCCCGA GGCCAAGGCC AGCTGCTACG GG - #CAGATGGA       1020                                                                          - GAGGCCAGAG GTCCCGATGC ACACCTTGCA CCCATTTATG GTCAATGTTA CA - #TGGGATGG       1080                                                                          - CAAAGACTTA TCCTTCACTG AGGAAGGCTA CCAGGTGCAC CCCAGGCTGG TG - #GTGATTGT       1140                                                                          - GCTGAACAAA GACCGGGAAT GGGAAAAGGT GGCCAAGTGG AAGAACAATA CG - #CTGAGCCT       1200                                                                          - GAGCCACGCC GTGTGGCCCA GGTACAAGTC CTTCTCCGAC TGTGAGCCGG AT - #GACAACCA       1260                                                                          - TCTCAGCATC GTCACCCTGG AGGAGGCCCC ATTCGTCATC GTGGAAGACA TA - #GACCCCCT       1320                                                                          - GACCGAGACG TGTGTGAGGA ACACCGTGCC ATGTCGGAAG TTCGTCAAAA TC - #AACAATTC       1380                                                                          - AACCAATGAG GGGATGAATG TGAAGAAATG CTGCAAGGGG TTCTGCATTG AT - #ATTCTGAA       1440                                                                          - GAAGCTTTCC AGAACTGTGA AGTTTACTTA CGACCTCTAT CTGGTGACCA AT - #GGGAAGCA       1500                                                                          - TGGCAAGAAA GTTAACAATG TGTGGAATGG AATGATCGGT GAAGTGGTCT AT - #CAACGGGC       1560                                                                          - AGTCATGGCA GTTGGCTCGC TCACCATCAA TGAGGAACGT TCTGAAGTGG TG - #GACTTCTC       1620                                                                          - TGTGCCCTTT GTGGAAACGG GAATCAGTGT CATGGTTTCA AGAAGTAATG GC - #ACCGTCTC       1680                                                                          - ACCTTCTGCT TTTCTAGAAC CATTCAGCGC CTCTGTCTGG GTGATGATGT TT - #GTGATGCT       1740                                                                          - GCTCATTGTT TCTGCCATAG CTGTTTTTGT CTTTGAATAC TTCAGCCCTG TT - #GGATACAA       1800                                                                          - CAGAAACTTA GCCAAAGGGA AAGCACCCCA TGGGCCTTCT TTTACAATTG GA - #AAAGCTAT       1860                                                                          - ATGGCTTCTT TGGGGCCTGG TGTTCAATAA CTCCGTGCCT GTCCAGAATC CT - #AAAGGGAC       1920                                                                          - CACCAGCAAG ATCATGGTAT CTGTATGGGC CTTCTTCGCT GTCATATTCC TG - #GCTAGCTA       1980                                                                          - CACAGCCAAT CTGGCTGCCT TCATGATCCA AGAGGAATTT GTGGACCAAG TG - #ACCGGCCT       2040                                                                          - CAGTGACAAA AAGTTTCAGA GACCTCATGA CTATTCCCCA CCTTTTCGAT TT - #GGGACAGT       2100                                                                          - GCCTAATGGA AGCACGGAGA GAAACATTCG GAATAACTAT CCCTACATGC AT - #CAGTACAT       2160                                                                          - GACCAAATTT AATCAGAAAG GAGTAGAGGA CGCCTTGGTC AGCCTGAAAA CG - #GGGAAGCT       2220                                                                          - GGACGCTTTC ATCTACGATG CCGCAGTCTT GAATTACAAG GCTGGGAGGG AT - #GAAGGCTG       2280                                                                          - CAAACTGGTG ACCATCGGGA GTGGGTACAT CTTTGCCACC ACCAGTTATG GA - #ATTGCCCT       2340                                                                          - TCAGAAAGGC TCTCCTTGGA AGAGGCAGAT CGACCTGGCC TTGCTTCAGT TT - #GTGGGTGA       2400                                                                          - TGGTGAGATG GAGGAGCTGG AGACCCTGTG GCTCACTGGG ATCGTCCACA AC - #GAGAAGAA       2460                                                                          - CGAGGTGATG AGCAACCAGC TGGACATTGA CAACATGGCG GGCGTATTCT AC - #ATGCTGGC       2520                                                                          - TGCCGCCATG GCCCTTAGCC TCATCACCTT CATCTGGGAG CACCTCTTCT AC - #TGGAAGCT       2580                                                                          - GCGCTTCTGT TTCACGGGCG TGTGCTCCGA CCGGCCTGGG TTGCTCTTCT CC - #ATCAGCAG       2640                                                                          - GGGCATCTAC AGCTGCATTC ATGGAGTGCA CATTGAAGAA AAGAAGAAGT CT - #CCAGACTT       2700                                                                          - CAATCTGACG GGATCCCAGA GCAACATGTT AAAACTCCTC CGGTCAGCCA AA - #AACATTTC       2760                                                                          - CAGCATGTCC AACATGAACT CCTCAAGAAT GGACTCACCC AAAAGAGCTG CT - #GACTTCAT       2820                                                                          - CCAAAGAGGT TCCCTCATCA TGGACATGGT TTCAGATAAG GGGAATTTGA TG - #TACTCAGA       2880                                                                          - CAACAGGTCC TTTCAGGGGA AAGAGAGCAT TTTTGGAGAC AACATGAACG AA - #CTCCAAAC       2940                                                                          - ATTTGTGGCC AACCGGCAGA AGGATAACCT CAATAACTAT GTATTCCAGG GA - #CAACATCC       3000                                                                          - TCTTACTCTC AATGAGTCCA ACCCTAACAC GGTGGAGGTG GCCGTGAGCA CA - #GAATCCAA       3060                                                                          - AGCGAACTCT AGACCCCGGC AGCTGTGGAA GAAATCCGTG GATTCCATAC GC - #CAGGATTC       3120                                                                          - ACTATCCCAG AATCCAGTCT CCCAGAGGGA TGAGGCAACA GCAGAGAATA GG - #ACCCACTC       3180                                                                          - CCTAAAGAGC CCTAGGTATC TTCCAGAAGA GATGGCCCAC TCTGACATTT CA - #GAAACGTC       3240                                                                          - AAATCGGGCC ACGTGCCACA GGGAACCTGA CAACAGTAAG AACCACAAAA CC - #AAGGACAA       3300                                                                          - CTTTAAAAGG TCAGTGGCCT CCAAATACCC CAAGGACTGT AGTGAGGTCG AG - #CGCACCTA       3360                                                                          - CCTGAAAACC AAATCAAGCT CCCCTAGAGA CAAGATCTAC ACTATAGATG GT - #GAGAAGGA       3420                                                                          - GCCTGGTTTC CACTTAGATC CACCCCAGTT TGTTGAAAAT GTGACCCTGC CC - #GAGAACGT       3480                                                                          - GGACTTCCCG GACCCCTACC AGGATCCCAG TGAAAACTTC CGCAAGGGGG AC - #TCCACGCT       3540                                                                          - GCCAATGAAC CGGAACCCCT TGCATAATGA AGAGGGGCTT TCCAACAACG AC - #CAGTATAA       3600                                                                          - ACTCTACTCC AAGCACTTCA CCTTGAAAGA CAAGGGTTCC CCGCACAGTG AG - #ACCAGCGA       3660                                                                          - GCGATACCGG CAGAACTCCA CGCACTGCAG AAGCTGCCTT TCCAACATGC CC - #ACCTATTC       3720                                                                          - AGGCCACTTC ACCATGAGGT CCCCCTTCAA GTGCGATGCC TGCCTGCGGA TG - #GGGAACCT       3780                                                                          - CTATGACATC GATGAAGACC AGATGCTTCA GGAGACAGGT AACCCAGCCA CC - #GGGGAGGA       3840                                                                          - GGTCTACCAG CAGGACTGGG CACAGAACAA TGCCCTTCAA TTACAAAAGA AC - #AAGCTAAG       3900                                                                          - GATTAGCCGT CAGCATTCCT ACGATAACAT TGTCGACAAA CCTAGGGAGC TA - #GACCTTAG       3960                                                                          - CAGGCCCTCC CGGAGCATAA GCCTCAAGGA CAGGGAACGG CTTCTGGAGG GA - #AATTTTTA       4020                                                                          - CGGCAGCCTG TTTAGTGTCC CCTCAAGCAA ACTCTCGGGG AAAAAAAGCT CC - #CTTTTCCC       4080                                                                          - CCAAGGTCTG GAGGACAGCA AGAGGAGCAA GTCTCTCTTG CCAGACCACA CC - #TCCGATAA       4140                                                                          - CCCTTTCCTC CACTCCCACA GGGATGACCA ACGCTTGGGT ATTGGGAGAT GC - #CCCTCGGA       4200                                                                          - CCCTTACAAA CACTCGTTGC CATCCCAGGC GGTGAATGAC AGCTATCTTC GG - #TCGTCCTT       4260                                                                          - GAGGTCAACG GCATCGTACT GTTCCAGGGA CAGTCGGGGC CACAATGATG TG - #TATATTTC       4320                                                                          - GGAGCATGTT ATGCCTTATG CTGCAAATAA GAATAATATG TACTCTACCC CC - #AGGGTTTT       4380                                                                          - AAATTCCTGC AGCAATAGAC GCGTGTACAA GAAAATGCCT AGTATCGAAT CT - #GATGTTTA       4440                                                                          - AAAATCTTCC ATTAATGTTT TATCTATAGG GAAATATACG TAATGGCCAA TG - #TTCTGGAG       4500                                                                          - GGTAAATGTT GAATGTCCAA TAGTGCCCTG CTAAGAGGAA GAAGATGTAG GG - #AGGTATTT       4560                                                                          - TGTTGTTGTT GTTGTTGGCT CTTTTGCACA CGGCTTCATG CCATAATCTT CC - #ACTCAAGG       4620                                                                          - AATCTTGTGA GGTGTGTGCT GAGCATGGCA GACACCAGAT AGGTGAGTCC TT - #AACCAAAA       4680                                                                          - ATAACTAACT ACATAAGGGC AAGTCTCCGG GACATGCCTA CTGGGTATGT TG - #CCAATAAT       4740                                                                          - GATGCATTGG ATGCCAATGG TGATGTTATG ATTTCCTATA TTCCAAATTC CA - #TTAAGGTC       4800                                                                          - AGCCCACCAT GTAATTTTCT CATCAGAAAT GCCTAATGGT TTCTCTAATA CA - #GAATAA         4858                                                                          - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1464 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Met Gly Arg Val Gly Tyr Trp Thr Leu Leu Va - #l Leu Pro Ala Leu Leu         #                15                                                           - Val Trp Arg Gly Pro Ala Pro Ser Ala Ala Al - #a Glu Lys Gly Pro Pro         #            30                                                               - Ala Leu Asn Ile Ala Val Met Leu Gly His Se - #r His Asp Val Thr Glu         #        45                                                                   - Arg Glu Leu Arg Thr Leu Trp Gly Pro Glu Gl - #n Ala Gly Gly Leu Pro         #    60                                                                       - Leu Asp Val Asn Val Val Ala Leu Leu Met As - #n Arg Thr Asp Pro Lys         #80                                                                           - Ser Leu Ile Thr His Val Cys Asp Leu Met Se - #r Gly Ala Arg Ile His         #                95                                                           - Gly Leu Val Phe Gly Asp Asp Thr Asp Gln Gl - #u Val Val Ala Gln Met         #           110                                                               - Leu Asp Phe Ile Ser Ser His Thr Phe Val Pr - #o Ile Leu Gly Ile His         #       125                                                                   - Gly Gly Ala Ser Met Ile Met Ala Asp Lys As - #p Pro Thr Ser Thr Phe         #   140                                                                       - Phe Gln Phe Gly Ala Ser Ile Gln Gln Gln Al - #a Thr Val Met Leu Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Met Gln Asp Tyr Asp Trp His Val Phe Se - #r Leu Val Thr Thr Ile         #               175                                                           - Phe Pro Gly Tyr Arg Glu Phe Ile Ser Phe Va - #l Lys Thr Thr Val Asp         #           190                                                               - Asn Ser Phe Val Gly Trp Asp Met Gln Asn Va - #l Ile Thr Leu Asp Thr         #       205                                                                   - Ser Phe Glu Asp Ala Lys Thr Gln Val Gln Le - #u Lys Lys Ile His Ser         #   220                                                                       - Ser Val Ile Leu Leu Tyr Cys Ser Lys Asp Gl - #u Ala Val Leu Ile Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ser Glu Ala Arg Ser Leu Gly Val Thr Gly Ty - #r Asp Phe Phe Trp Ile         #               255                                                           - Val Pro Ser Leu Val Ser Gly Asn Thr Glu Le - #u Ile Pro Lys Glu Phe         #           270                                                               - Pro Ser Gly Leu Ile Ser Val Ser Tyr Asp As - #p Trp Asp Tyr Ser Leu         #       285                                                                   - Glu Ala Arg Val Arg Asp Gly Ile Gly Ile Le - #u Thr Thr Ala Ala Ser         #   300                                                                       - Ser Met Leu Glu Lys Phe Ser Tyr Ile Pro Gl - #u Ala Lys Ala Ser Cys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Tyr Gly Gln Met Glu Arg Pro Glu Val Pro Me - #t His Thr Leu His Pro         #               335                                                           - Phe Met Val Asn Val Thr Trp Asp Gly Lys As - #p Leu Ser Phe Thr Glu         #           350                                                               - Glu Gly Tyr Gln Val His Pro Arg Leu Val Va - #l Ile Val Leu Asn Lys         #       365                                                                   - Asp Arg Glu Trp Glu Lys Val Ala Lys Trp Ly - #s Asn Asn Thr Leu Ser         #   380                                                                       - Leu Ser His Ala Val Trp Pro Arg Tyr Lys Se - #r Phe Ser Asp Cys Glu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Pro Asp Asp Asn His Leu Ser Ile Val Thr Le - #u Glu Glu Ala Pro Phe         #               415                                                           - Val Ile Val Glu Asp Ile Asp Pro Leu Thr Gl - #u Thr Cys Val Arg Asn         #           430                                                               - Thr Val Pro Cys Arg Lys Phe Val Lys Ile As - #n Asn Ser Thr Asn Glu         #       445                                                                   - Gly Met Asn Val Lys Lys Cys Cys Lys Gly Ph - #e Cys Ile Asp Ile Leu         #   460                                                                       - Lys Lys Leu Ser Arg Thr Val Lys Phe Thr Ty - #r Asp Leu Tyr Leu Val         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Thr Asn Gly Lys His Gly Lys Lys Val Asn As - #n Val Trp Asn Gly Met         #               495                                                           - Ile Gly Glu Val Val Tyr Gln Arg Ala Val Me - #t Ala Val Gly Ser Leu         #           510                                                               - Thr Ile Asn Glu Glu Arg Ser Glu Val Val As - #p Phe Ser Val Pro Phe         #       525                                                                   - Val Glu Thr Gly Ile Ser Val Met Val Ser Ar - #g Ser Asn Gly Thr Val         #   540                                                                       - Ser Pro Ser Ala Phe Leu Glu Pro Phe Ser Al - #a Ser Val Trp Val Met         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Met Phe Val Met Leu Leu Ile Val Ser Ala Il - #e Ala Val Phe Val Phe         #               575                                                           - Glu Tyr Phe Ser Pro Val Gly Tyr Asn Arg As - #n Leu Ala Lys Gly Lys         #           590                                                               - Ala Pro His Gly Pro Ser Phe Thr Ile Gly Ly - #s Ala Ile Trp Leu Leu         #       605                                                                   - Trp Gly Leu Val Phe Asn Asn Ser Val Pro Va - #l Gln Asn Pro Lys Gly         #   620                                                                       - Thr Thr Ser Lys Ile Met Val Ser Val Trp Al - #a Phe Phe Ala Val Ile         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Phe Leu Ala Ser Tyr Thr Ala Asn Leu Ala Al - #a Phe Met Ile Gln Glu         #               655                                                           - Glu Phe Val Asp Gln Val Thr Gly Leu Ser As - #p Lys Lys Phe Gln Arg         #           670                                                               - Pro His Asp Tyr Ser Pro Pro Phe Arg Phe Gl - #y Thr Val Pro Asn Gly         #       685                                                                   - Ser Thr Glu Arg Asn Ile Arg Asn Asn Tyr Pr - #o Tyr Met His Gln Tyr         #   700                                                                       - Met Thr Lys Phe Asn Gln Lys Gly Val Glu As - #p Ala Leu Val Ser Leu         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Lys Thr Gly Lys Leu Asp Ala Phe Ile Tyr As - #p Ala Ala Val Leu Asn         #               735                                                           - Tyr Lys Ala Gly Arg Asp Glu Gly Cys Lys Le - #u Val Thr Ile Gly Ser         #           750                                                               - Gly Tyr Ile Phe Ala Thr Thr Ser Tyr Gly Il - #e Ala Leu Gln Lys Gly         #       765                                                                   - Ser Pro Trp Lys Arg Gln Ile Asp Leu Ala Le - #u Leu Gln Phe Val Gly         #   780                                                                       - Asp Gly Glu Met Glu Glu Leu Glu Thr Leu Tr - #p Leu Thr Gly Ile Val         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - His Asn Glu Lys Asn Glu Val Met Ser Asn Gl - #n Leu Asp Ile Asp Asn         #               815                                                           - Met Ala Gly Val Phe Tyr Met Leu Ala Ala Al - #a Met Ala Leu Ser Leu         #           830                                                               - Ile Thr Phe Ile Trp Glu His Leu Phe Tyr Tr - #p Lys Leu Arg Phe Cys         #       845                                                                   - Phe Thr Gly Val Cys Ser Asp Arg Pro Gly Le - #u Leu Phe Ser Ile Ser         #   860                                                                       - Arg Gly Ile Tyr Ser Cys Ile His Gly Val Hi - #s Ile Glu Glu Lys Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Lys Ser Pro Asp Phe Asn Leu Thr Gly Ser Gl - #n Ser Asn Met Leu Lys         #               895                                                           - Leu Leu Arg Ser Ala Lys Asn Ile Ser Ser Me - #t Ser Asn Met Asn Ser         #           910                                                               - Ser Arg Met Asp Ser Pro Lys Arg Ala Ala As - #p Phe Ile Gln Arg Gly         #       925                                                                   - Ser Leu Ile Met Asp Met Val Ser Asp Lys Gl - #y Asn Leu Met Tyr Ser         #   940                                                                       - Asp Asn Arg Ser Phe Gln Gly Lys Glu Ser Il - #e Phe Gly Asp Asn Met         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Asn Glu Leu Gln Thr Phe Val Ala Asn Arg Gl - #n Lys Asp Asn Leu Asn         #               975                                                           - Asn Tyr Val Phe Gln Gly Gln His Pro Leu Th - #r Leu Asn Glu Ser Asn         #           990                                                               - Pro Asn Thr Val Glu Val Ala Val Ser Thr Gl - #u Ser Lys Ala Asn Ser         #      10050                                                                  - Arg Pro Arg Gln Leu Trp Lys Lys Ser Val As - #p Ser Ile Arg Gln Asp         #  10205                                                                      - Ser Leu Ser Gln Asn Pro Val Ser Gln Arg As - #p Glu Ala Thr Ala Glu         #               10401030 - #                1035                              - Asn Arg Thr His Ser Leu Lys Ser Pro Arg Ty - #r Leu Pro Glu Glu Met         #              10550                                                          - Ala His Ser Asp Ile Ser Glu Thr Ser Asn Ar - #g Ala Thr Cys His Arg         #          10705                                                              - Glu Pro Asp Asn Ser Lys Asn His Lys Thr Ly - #s Asp Asn Phe Lys Arg         #      10850                                                                  - Ser Val Ala Ser Lys Tyr Pro Lys Asp Cys Se - #r Glu Val Glu Arg Thr         #  11005                                                                      - Tyr Leu Lys Thr Lys Ser Ser Ser Pro Arg As - #p Lys Ile Tyr Thr Ile         #               11201110 - #                1115                              - Asp Gly Glu Lys Glu Pro Gly Phe His Leu As - #p Pro Pro Gln Phe Val         #              11350                                                          - Glu Asn Val Thr Leu Pro Glu Asn Val Asp Ph - #e Pro Asp Pro Tyr Gln         #          11505                                                              - Asp Pro Ser Glu Asn Phe Arg Lys Gly Asp Se - #r Thr Leu Pro Met Asn         #      11650                                                                  - Arg Asn Pro Leu His Asn Glu Glu Gly Leu Se - #r Asn Asn Asp Gln Tyr         #  11805                                                                      - Lys Leu Tyr Ser Lys His Phe Thr Leu Lys As - #p Lys Gly Ser Pro His         #               12001190 - #                1195                              - Ser Glu Thr Ser Glu Arg Tyr Arg Gln Asn Se - #r Thr His Cys Arg Ser         #              12150                                                          - Cys Leu Ser Asn Met Pro Thr Tyr Ser Gly Hi - #s Phe Thr Met Arg Ser         #          12305                                                              - Pro Phe Lys Cys Asp Ala Cys Leu Arg Met Gl - #y Asn Leu Tyr Asp Ile         #      12450                                                                  - Asp Glu Asp Gln Met Leu Gln Glu Thr Gly As - #n Pro Ala Thr Gly Glu         #  12605                                                                      - Glu Val Tyr Gln Gln Asp Trp Ala Gln Asn As - #n Ala Leu Gln Leu Gln         #               12801270 - #                1275                              - Lys Asn Lys Leu Arg Ile Ser Arg Gln His Se - #r Tyr Asp Asn Ile Val         #              12950                                                          - Asp Lys Pro Arg Glu Leu Asp Leu Ser Arg Pr - #o Ser Arg Ser Ile Ser         #          13105                                                              - Leu Lys Asp Arg Glu Arg Leu Leu Glu Gly As - #n Phe Tyr Gly Ser Leu         #      13250                                                                  - Phe Ser Val Pro Ser Ser Lys Leu Ser Gly Ly - #s Lys Ser Ser Leu Phe         #  13405                                                                      - Pro Gln Gly Leu Glu Asp Ser Lys Arg Ser Ly - #s Ser Leu Leu Pro Asp         #               13601350 - #                1355                              - His Thr Ser Asp Asn Pro Phe Leu His Ser Hi - #s Arg Asp Asp Gln Arg         #              13750                                                          - Leu Gly Ile Gly Arg Cys Pro Ser Asp Pro Ty - #r Lys His Ser Leu Pro         #          13905                                                              - Ser Gln Ala Val Asn Asp Ser Tyr Leu Arg Se - #r Ser Leu Arg Ser Thr         #      14050                                                                  - Ala Ser Tyr Cys Ser Arg Asp Ser Arg Gly Hi - #s Asn Asp Val Tyr Ile         #  14205                                                                      - Ser Glu His Val Met Pro Tyr Ala Ala Asn Ly - #s Asn Asn Met Tyr Ser         #               14401430 - #                1435                              - Thr Pro Arg Val Leu Asn Ser Cys Ser Asn Ar - #g Arg Val Tyr Lys Lys         #              14550                                                          - Met Pro Ser Ile Glu Ser Asp Val                                                         1460                                                              - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2916 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - GCCGCGCAGA GCCAGGCCCG CGGCCCGAGC CCATGAGCAC CATGCGCCTG TT - #GACGCTCG         60                                                                          - CCCTGCTGTT CTCCTGCTCC CTCGCCCGTG CCGCGTGCGA CCCCAAGATC GT - #CAACATTG        120                                                                          - GCGCGGTGCT GAGCACGCGG AAGCACGAGC AGATGTTCCG CGAGGCCGTG AA - #CCAGGCCA        180                                                                          - ACAAGCGGCA CGGCTCCTGG AAGATTCAGC TCAATGCCAC CTCCGTCACG CA - #CAAGCCCA        240                                                                          - ACGCCATCCA GATGGCTCTG TCGGTGTGCG AGGACCTCAT CTCCAGCCAG GT - #CTACGCCA        300                                                                          - TCCTAGTTAG CCATCCACCT ACCCCCAACG ACCACTTCAC TCCCACCCCT GT - #CTCCTACA        360                                                                          - CAGCCGGCTT CTACCGCATA CCCGTGCTGG GGCTGACCAC CCGCATGTCC AT - #CTACTCGG        420                                                                          - ACAAGAGCAT CCACCTGAGC TTCCTGCGCA CCGTGCCGCC CTACTCCCAC CA - #GTCCAGCG        480                                                                          - TGTGGTTTGA GATGATGCGT GTCTACAGCT GGAACCACAT CATCCTGCTG GT - #CAGCGACG        540                                                                          - ACCACGAGGG CCGGGCGGCT CAGAAACGCC TGGAGACGCT GCTGGAGGAG CG - #TGAGTCCA        600                                                                          - AGGCAGAGAA GGTGCTGCAG TTTGACCCAG GGACCAAGAA CGTGACGGCC CT - #GCTGATGG        660                                                                          - AGGCGAAAGA GCTGGAGGCC CGGGTCATCA TCCTTTCTGC CAGCGAGGAC GA - #TGCTGCCA        720                                                                          - CTGTATACCG CGCAGCCGCG ATGCTGAACA TGACGGGCTC CGGGTACGTG TG - #GCTGGTCG        780                                                                          - GCGAGCGCGA GATCTCGGGG AACGCCCTGG CCTACGCCCC AGACGGCATC CT - #CGGGCTGC        840                                                                          - AGCTCATCAA CGGCAAGAAC GAGTCGGCCC ACATCAGCGA CGCCGTGGGC GT - #GGTGGCCC        900                                                                          - AGGCCGTGCA CGAGCTCCTC GAGAAGGAGA ACATCACCGA CCCGCCGCGG GG - #CTGCGTGG        960                                                                          - GCAACACCAA CATCTGGAAG ACCGGGCCGC TCTTCAAGAG AGTGCTGATG TC - #TTCCAAGT       1020                                                                          - ATGCGGATGG GGTGACTGGT CGCGTGGAGT TCAATGAGGA TGGGGACCGG AA - #GTTCGCCA       1080                                                                          - ACTACAGCAT CATGAACCTG CAGAACCGCA AGCTGGTGCA AGTGGGCATC TA - #CAATGGCA       1140                                                                          - CCCACGTCAT CCCTAATGAC AGGAAGATCA TCTGGCCAGG CGGAGAGACA GA - #GAAGCCTC       1200                                                                          - GAGGGTACCA GATGTCCACC AGACTGAAGA TTGTGACGAT CCACCAGGAG CC - #CTTCGTGT       1260                                                                          - ACGTCAAGCC CACGCTGAGT GATGGGACAT GCAAGGAGGA GTTCACAGTC AA - #CGGCGACC       1320                                                                          - CAGTCAAGAA GGTGATCTGC ACCGGGCCCA ACGACACGTC GCCGGGCAGC CC - #CCGCCACA       1380                                                                          - CGGTGCCTCA GTGTTGCTAC GGCTTTTGCA TCGACCTGCT CATCAAGCTG GC - #ACGGACCA       1440                                                                          - TGAACTTCAC CTACGAGGTG CACCTGGTGG CAGATGGCAA GTTCGGCACA CA - #GGAGCGGG       1500                                                                          - TGAACAACAG CAACAAGAAG GAGTGGAATG GGATGATGGG CGAGCTGCTC AG - #CGGGCAGG       1560                                                                          - CAGACATGAT CGTGGCGCCG CTAACCATAA ACAACGAGCG CGCGCAGTAC AT - #CGAGTTTT       1620                                                                          - CCAAGCCCTT CAAGTACCAG GGCCTGACTA TTCTGGTCAA GAAGGAGATT CC - #CCGGAGCA       1680                                                                          - CGCTGGACTC GTTCATGCAG CCGTTCCAGA GCACACTGTG GCTGCTGGTG GG - #GCTGTCGG       1740                                                                          - TGCACGTGGT GGCCGTGATG CTGTACCTGC TGGACCGCTT CAGCCCCTTC GG - #CCGGTTCA       1800                                                                          - AGGTGAACAG CGAGGAGGAG GAGGAGGACG CACTGACCCT GTCCTCGGCC AT - #GTGGTTCT       1860                                                                          - CCTGGGGCGT CCTGCTCAAC TCCGGCATCG GGGAAGGCGC CCCCAGAAGC TT - #CTCAGCGC       1920                                                                          - GCATCCTGGG CATGGTGTGG GCCGGCTTTG CCATGATCAT CGTGGCCTCC TA - #CACTGCCA       1980                                                                          - ACTTGGCGGC CTTCCTGGTG CTGGACCGGC CGGAGGAGCG CATCACGGGC AT - #CAACGACC       2040                                                                          - CTCGGCTGAG GAACCCCTCG GACAAGTTTA TCTACGCCAC GGTGAAGCAG AG - #CTCCGTGG       2100                                                                          - ATATCTACTT CCGGCGCCAG GTGGAGCTGA GCACCATGTA CCGGCATATG GA - #GAAGCACA       2160                                                                          - ACTACGAGAG TGCGGCGGAG GCCATCCAGG CCGTGAGAGA CAACAAGCTG CA - #TGCCTTCA       2220                                                                          - TCTGGGACTC GGCGGTGCTG GAGTTCGAGG CCTCGCAGAA GTGCGACCTG GT - #GACGACTG       2280                                                                          - GAGAGCTGTT TTTCCGCTCG GGCTTCGGCA TAGGCATGCG CAAAGACAGC CC - #CTGGAAGC       2340                                                                          - AGAACGTCTC CCTGTCCATC CTCAAGTCCC ACGAGAATGG CTTCATGGAA GA - #CCTGGACA       2400                                                                          - AGACGTGGGT TCGGTATCAG GAATGTGACT CGCGCAGCAA CGCCCCTGCA AC - #CCTTACTT       2460                                                                          - TTGAGAACAT GGCCGGGGTC TTCATGCTGG TAGCTGGGGG CATCGTGGCC GG - #GATCTTCC       2520                                                                          - TGATTTTCAT CGAGATTGCC TACAAGCGGC ACAAGGATGC TCGCCGGAAG CA - #GATGCAGC       2580                                                                          - TGGCCTTTGC CGCCGTTAAC GTGTGGCGGA AGAACCTGCA GGATAGAAAG AG - #TGGTAGAG       2640                                                                          - CAGAGCCTGA CCCTAAAAAG AAAGCCACAT TTAGGGCTAT CACCTCCACC CT - #GGCTTCCA       2700                                                                          - GCTTCAAGAG GCGTAGGTCC TCCAAAGACA CGAGCACCGG GGGTGGACGC GG - #CGCTTTGC       2760                                                                          - AAAACCAAAA AGACACAGTG CTGCCGCGAC GGCCTATTGA GAGGGAGGAG GG - #CCAGCTGC       2820                                                                          - AGCTGTGTTC CCGTCATAGG GAGAGCTGAG ACTCCCCGCC CGCCCTCCTC TG - #CCCCCCTC       2880                                                                          #     2916         ACAG ACGGACGGGA CAGCGG                                     - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2835 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - GCCGCGCAGA GCCAGGCCCG CGGCCCGAGC CCATGAGCAC CATGCGCCTG TT - #GACGCTCG         60                                                                          - CCCTGCTGTT CTCCTGCTCC CTCGCCCGTG CCGCGTGCGA CCCCAAGATC GT - #CAACATTG        120                                                                          - GCGCGGTGCT GAGCACGCGG AAGCACGAGC AGATGTTCCG CGAGGCCGTG AA - #CCAGGCCA        180                                                                          - ACAAGCGGCA CGGCTCCTGG AAGATTCAGC TCAATGCCAC CTCCGTCACG CA - #CAAGCCCA        240                                                                          - ACGCCATCCA GATGGCTCTG TCGGTGTGCG AGGACCTCAT CTCCAGCCAG GT - #CTACGCCA        300                                                                          - TCCTAGTTAG CCATCCACCT ACCCCCAACG ACCACTTCAC TCCCACCCCT GT - #CTCCTACA        360                                                                          - CAGCCGGCTT CTACCGCATA CCCGTGCTGG GGCTGACCAC CCGCATGTCC AT - #CTACTCGG        420                                                                          - ACAAGAGCAT CCACCTGAGC TTCCTGCGCA CCGTGCCGCC CTACTCCCAC CA - #GTCCAGCG        480                                                                          - TGTGGTTTGA GATGATGCGT GTCTACAGCT GGAACCACAT CATCCTGCTG GT - #CAGCGACG        540                                                                          - ACCACGAGGG CCGGGCGGCT CAGAAACGCC TGGAGACGCT GCTGGAGGAG CG - #TGAGTCCA        600                                                                          - AGGCAGAGAA GGTGCTGCAG TTTGACCCAG GGACCAAGAA CGTGACGGCC CT - #GCTGATGG        660                                                                          - AGGCGAAAGA GCTGGAGGCC CGGGTCATCA TCCTTTCTGC CAGCGAGGAC GA - #TGCTGCCA        720                                                                          - CTGTATACCG CGCAGCCGCG ATGCTGAACA TGACGGGCTC CGGGTACGTG TG - #GCTGGTCG        780                                                                          - GCGAGCGCGA GATCTCGGGG AACGCCCTGG CCTACGCCCC AGACGGCATC CT - #CGGGCTGC        840                                                                          - AGCTCATCAA CGGCAAGAAC GAGTCGGCCC ACATCAGCGA CGCCGTGGGC GT - #GGTGGCCC        900                                                                          - AGGCCGTGCA CGAGCTCCTC GAGAAGGAGA ACATCACCGA CCCGCCGCGG GG - #CTGCGTGG        960                                                                          - GCAACACCAA CATCTGGAAG ACCGGGCCGC TCTTCAAGAG AGTGCTGATG TC - #TTCCAAGT       1020                                                                          - ATGCGGATGG GGTGACTGGT CGCGTGGAGT TCAATGAGGA TGGGGACCGG AA - #GTTCGCCA       1080                                                                          - ACTACAGCAT CATGAACCTG CAGAACCGCA AGCTGGTGCA AGTGGGCATC TA - #CAATGGCA       1140                                                                          - CCCACGTCAT CCCTAATGAC AGGAAGATCA TCTGGCCAGG CGGAGAGACA GA - #GAAGCCTC       1200                                                                          - GAGGGTACCA GATGTCCACC AGACTGAAGA TTGTGACGAT CCACCAGGAG CC - #CTTCGTGT       1260                                                                          - ACGTCAAGCC CACGCTGAGT GATGGGACAT GCAAGGAGGA GTTCACAGTC AA - #CGGCGACC       1320                                                                          - CAGTCAAGAA GGTGATCTGC ACCGGGCCCA ACGACACGTC GCCGGGCAGC CC - #CCGCCACA       1380                                                                          - CGGTGCCTCA GTGTTGCTAC GGCTTTTGCA TCGACCTGCT CATCAAGCTG GC - #ACGGACCA       1440                                                                          - TGAACTTCAC CTACGAGGTG CACCTGGTGG CAGATGGCAA GTTCGGCACA CA - #GGAGCGGG       1500                                                                          - TGAACAACAG CAACAAGAAG GAGTGGAATG GGATGATGGG CGAGCTGCTC AG - #CGGGCAGG       1560                                                                          - CAGACATGAT CGTGGCGCCG CTAACCATAA ACAACGAGCG CGCGCAGTAC AT - #CGAGTTTT       1620                                                                          - CCAAGCCCTT CAAGTACCAG GGCCTGACTA TTCTGGTCAA GAAGGAGATT CC - #CCGGAGCA       1680                                                                          - CGCTGGACTC GTTCATGCAG CCGTTCCAGA GCACACTGTG GCTGCTGGTG GG - #GCTGTCGG       1740                                                                          - TGCACGTGGT GGCCGTGATG CTGTACCTGC TGGACCGCTT CAGCCCCTTC GG - #CCGGTTCA       1800                                                                          - AGGTGAACAG CGAGGAGGAG GAGGAGGACG CACTGACCCT GTCCTCGGCC AT - #GTGGTTCT       1860                                                                          - CCTGGGGCGT CCTGCTCAAC TCCGGCATCG GGGAAGGCGC CCCCAGAAGC TT - #CTCAGCGC       1920                                                                          - GCATCCTGGG CATGGTGTGG GCCGGCTTTG CCATGATCAT CGTGGCCTCC TA - #CACTGCCA       1980                                                                          - ACTTGGCGGC CTTCCTGGTG CTGGACCGGC CGGAGGAGCG CATCACGGGC AT - #CAACGACC       2040                                                                          - CTCGGCTGAG GAACCCCTCG GACAAGTTTA TCTACGCCAC GGTGAAGCAG AG - #CTCCGTGG       2100                                                                          - ATATCTACTT CCGGCGCCAG GTGGAGCTGA GCACCATGTA CCGGCATATG GA - #GAAGCACA       2160                                                                          - ACTACGAGAG TGCGGCGGAG GCCATCCAGG CCGTGAGAGA CAACAAGCTG CA - #TGCCTTCA       2220                                                                          - TCTGGGACTC GGCGGTGCTG GAGTTCGAGG CCTCGCAGAA GTGCGACCTG GT - #GACGACTG       2280                                                                          - GAGAGCTGTT TTTCCGCTCG GGCTTCGGCA TAGGCATGCG CAAAGACAGC CC - #CTGGAAGC       2340                                                                          - AGAACGTCTC CCTGTCCATC CTCAAGTCCC ACGAGAATGG CTTCATGGAA GA - #CCTGGACA       2400                                                                          - AGACGTGGGT TCGGTATCAG GAATGTGACT CGCGCAGCAA CGCCCCTGCA AC - #CCTTACTT       2460                                                                          - TTGAGAACAT GGCCGGGGTC TTCATGCTGG TAGCTGGGGG CATCGTGGCC GG - #GATCTTCC       2520                                                                          - TGATTTTCAT CGAGATTGCC TACAAGCGGC ACAAGGATGC TCGCCGGAAG CA - #GATGCAGC       2580                                                                          - TGGCCTTTGC CGCCGTTAAC GTGTGGCGGA AGAACCTGCA GGATAGAAAG AG - #TGGTAGAG       2640                                                                          - CAGAGCCTGA CCCTAAAAAG AAAGCCACAT TTAGGGCTAT CACCTCCACC CT - #GGCTTCCA       2700                                                                          - GCTTCAAGAG GCGTAGGTCC TCCAAAGACA CGCAGTACCA TCCCACTGAT AT - #CACGGGCC       2760                                                                          - CGCTCAACCT CTCAGATCCC TCGGTCAGCA CCGTGGTGTG AGGCCCCCGG AG - #CGCCCACC       2820                                                                          #  2835                                                                       - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2727 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - GCCGCGCAGA GCCAGGCCCG CGGCCCGAGC CCATGAGCAC CATGCGCCTG TT - #GACGCTCG         60                                                                          - CCCTGCTGTT CTCCTGCTCC CTCGCCCGTG CCGCGTGCGA CCCCAAGATC GT - #CAACATTG        120                                                                          - GCGCGGTGCT GAGCACGCGG AAGCACGAGC AGATGTTCCG CGAGGCCGTG AA - #CCAGGCCA        180                                                                          - ACAAGCGGCA CGGCTCCTGG AAGATTCAGC TCAATGCCAC CTCCGTCACG CA - #CAAGCCCA        240                                                                          - ACGCCATCCA GATGGCTCTG TCGGTGTGCG AGGACCTCAT CTCCAGCCAG GT - #CTACGCCA        300                                                                          - TCCTAGTTAG CCATCCACCT ACCCCCAACG ACCACTTCAC TCCCACCCCT GT - #CTCCTACA        360                                                                          - CAGCCGGCTT CTACCGCATA CCCGTGCTGG GGCTGACCAC CCGCATGTCC AT - #CTACTCGG        420                                                                          - ACAAGAGCAT CCACCTGAGC TTCCTGCGCA CCGTGCCGCC CTACTCCCAC CA - #GTCCAGCG        480                                                                          - TGTGGTTTGA GATGATGCGT GTCTACAGCT GGAACCACAT CATCCTGCTG GT - #CAGCGACG        540                                                                          - ACCACGAGGG CCGGGCGGCT CAGAAACGCC TGGAGACGCT GCTGGAGGAG CG - #TGAGTCCA        600                                                                          - AGGCAGAGAA GGTGCTGCAG TTTGACCCAG GGACCAAGAA CGTGACGGCC CT - #GCTGATGG        660                                                                          - AGGCGAAAGA GCTGGAGGCC CGGGTCATCA TCCTTTCTGC CAGCGAGGAC GA - #TGCTGCCA        720                                                                          - CTGTATACCG CGCAGCCGCG ATGCTGAACA TGACGGGCTC CGGGTACGTG TG - #GCTGGTCG        780                                                                          - GCGAGCGCGA GATCTCGGGG AACGCCCTGG CCTACGCCCC AGACGGCATC CT - #CGGGCTGC        840                                                                          - AGCTCATCAA CGGCAAGAAC GAGTCGGCCC ACATCAGCGA CGCCGTGGGC GT - #GGTGGCCC        900                                                                          - AGGCCGTGCA CGAGCTCCTC GAGAAGGAGA ACATCACCGA CCCGCCGCGG GG - #CTGCGTGG        960                                                                          - GCAACACCAA CATCTGGAAG ACCGGGCCGC TCTTCAAGAG AGTGCTGATG TC - #TTCCAAGT       1020                                                                          - ATGCGGATGG GGTGACTGGT CGCGTGGAGT TCAATGAGGA TGGGGACCGG AA - #GTTCGCCA       1080                                                                          - ACTACAGCAT CATGAACCTG CAGAACCGCA AGCTGGTGCA AGTGGGCATC TA - #CAATGGCA       1140                                                                          - CCCACGTCAT CCCTAATGAC AGGAAGATCA TCTGGCCAGG CGGAGAGACA GA - #GAAGCCTC       1200                                                                          - GAGGGTACCA GATGTCCACC AGACTGAAGA TTGTGACGAT CCACCAGGAG CC - #CTTCGTGT       1260                                                                          - ACGTCAAGCC CACGCTGAGT GATGGGACAT GCAAGGAGGA GTTCACAGTC AA - #CGGCGACC       1320                                                                          - CAGTCAAGAA GGTGATCTGC ACCGGGCCCA ACGACACGTC GCCGGGCAGC CC - #CCGCCACA       1380                                                                          - CGGTGCCTCA GTGTTGCTAC GGCTTTTGCA TCGACCTGCT CATCAAGCTG GC - #ACGGACCA       1440                                                                          - TGAACTTCAC CTACGAGGTG CACCTGGTGG CAGATGGCAA GTTCGGCACA CA - #GGAGCGGG       1500                                                                          - TGAACAACAG CAACAAGAAG GAGTGGAATG GGATGATGGG CGAGCTGCTC AG - #CGGGCAGG       1560                                                                          - CAGACATGAT CGTGGCGCCG CTAACCATAA ACAACGAGCG CGCGCAGTAC AT - #CGAGTTTT       1620                                                                          - CCAAGCCCTT CAAGTACCAG GGCCTGACTA TTCTGGTCAA GAAGGAGATT CC - #CCGGAGCA       1680                                                                          - CGCTGGACTC GTTCATGCAG CCGTTCCAGA GCACACTGTG GCTGCTGGTG GG - #GCTGTCGG       1740                                                                          - TGCACGTGGT GGCCGTGATG CTGTACCTGC TGGACCGCTT CAGCCCCTTC GG - #CCGGTTCA       1800                                                                          - AGGTGAACAG CGAGGAGGAG GAGGAGGACG CACTGACCCT GTCCTCGGCC AT - #GTGGTTCT       1860                                                                          - CCTGGGGCGT CCTGCTCAAC TCCGGCATCG GGGAAGGCGC CCCCAGAAGC TT - #CTCAGCGC       1920                                                                          - GCATCCTGGG CATGGTGTGG GCCGGCTTTG CCATGATCAT CGTGGCCTCC TA - #CACTGCCA       1980                                                                          - ACTTGGCGGC CTTCCTGGTG CTGGACCGGC CGGAGGAGCG CATCACGGGC AT - #CAACGACC       2040                                                                          - CTCGGCTGAG GAACCCCTCG GACAAGTTTA TCTACGCCAC GGTGAAGCAG AG - #CTCCGTGG       2100                                                                          - ATATCTACTT CCGGCGCCAG GTGGAGCTGA GCACCATGTA CCGGCATATG GA - #GAAGCACA       2160                                                                          - ACTACGAGAG TGCGGCGGAG GCCATCCAGG CCGTGAGAGA CAACAAGCTG CA - #TGCCTTCA       2220                                                                          - TCTGGGACTC GGCGGTGCTG GAGTTCGAGG CCTCGCAGAA GTGCGACCTG GT - #GACGACTG       2280                                                                          - GAGAGCTGTT TTTCCGCTCG GGCTTCGGCA TAGGCATGCG CAAAGACAGC CC - #CTGGAAGC       2340                                                                          - AGAACGTCTC CCTGTCCATC CTCAAGTCCC ACGAGAATGG CTTCATGGAA GA - #CCTGGACA       2400                                                                          - AGACGTGGGT TCGGTATCAG GAATGTGACT CGCGCAGCAA CGCCCCTGCA AC - #CCTTACTT       2460                                                                          - TTGAGAACAT GGCCGGGGTC TTCATGCTGG TAGCTGGGGG CATCGTGGCC GG - #GATCTTCC       2520                                                                          - TGATTTTCAT CGAGATTGCC TACAAGCGGC ACAAGGATGC TCGCCGGAAG CA - #GATGCAGC       2580                                                                          - TGGCCTTTGC CGCCGTTAAC GTGTGGCGGA AGAACCTGCA GCAGTACCAT CC - #CACTGATA       2640                                                                          - TCACGGGCCC GCTCAACCTC TCAGATCCCT CGGTCAGCAC CGTGGTGTGA GG - #CCCCCGGA       2700                                                                          #           2727   TTTA GCCCGGC                                               - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #           30     TTCC CTCAGACAAG                                            - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #             28   GCCA TGTTCTCA                                              - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #           30     CTGG CGCGATCCGG                                            - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #             28   CAAT AACGCCAC                                              - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #             28   TCAG CTAAAAAC                                              - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #           30     CCGG TATCGATCAC                                            - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #          32      ACCT TCTCGGGCTA CA                                         - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #             27   GACA GAAATGA                                               __________________________________________________________________________

What is claimed is:
 1. A recombinant DNA molecule having a nucleotidesequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:12 and SEQ ID NO
 13. 2. An expression vector comprisinga DNA molecule of claim
 1. 3. A recombinant cell line comprised of ahost cell transformed with an expression vector of claim
 2. 4. Arecombinantly produced NMDA receptor subunit, wherein said subunit isexpressed from an expression vector, wherein said vector comprises a DNAmolecule having a nucleotide sequence selected from the group consistingof SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.